FRET protease assays for clostridial toxins

ABSTRACT

The present invention provides clostridial toxin substrates useful in assaying for the protease activity of any clostridial toxin, including botulinum toxins of all serotypes as well as tetanus toxins. A clostridial toxin substrate of the invention contains a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a clostridial toxin recognition sequence that includes a cleavage site, where the cleavage site intervenes between the donor fluorophore and the acceptor and where, under the appropriate conditions, resonance energy transfer is exhibited between the donor fluorophore and the acceptor.

This application is a continuation and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 12/192,798, filed Oct.21, 2008, a divisional that claims priority pursuant to 35 U.S.C. §120to U.S. patent application Ser. No. 11/780,925, filed Jul. 20, 2007, nowabandoned, a divisional that claims priority pursuant to 35 U.S.C. §120to U.S. patent application Ser. No. 09/942,098, filed Aug. 28, 2001, nowU.S. Pat. No. 7,332,567, each of which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to fluorescence resonance energytransfer and protease assays, for example, assays for protease activityof clostridial toxins such botulinum toxins and tetanus toxins, and morespecifically, to intramolecularly quenched substrates and methods forassaying for clostridial toxin protease activity.

2. Background Information

The neuroparalytic syndrome of tetanus and the rare but potentiallyfatal disease, botulism, are caused by neurotoxins produced by bacteriaof the genus Clostridium. These clostridial neurotoxins are highlypotent and specific poisons of neural cells, with the human lethal doseof the botulinum toxins on the order of micrograms. Thus, the presenceof even minute levels of botulinum toxins in foodstuffs represents apublic health hazard that must be avoided through rigorous testing.

However, in spite of their potentially deleterious effects, lowcontrolled doses of botulinum neurotoxins have been successfully used astherapeutics. These toxins have been used in the therapeutic managementof a variety of focal and segmental dystonias, of strabismus and otherconditions in which a reversible depression of a cholinergic nerveterminal activity is desired. Established therapeutic uses of botulinumneurotoxins in humans include, for example, blepharospasm, hemifacialspasm, laringeal dysphonia, focal hyperhidrosis, hypersalivation,oromandibular dystonia, cervical dystonia, torticollis, strabismus,limbs dystonia, occupational cramps and myokymia (Rossetto et al,Toxicon 39:27-41 (2001)). Intramuscular injection of spastic tissue withsmall quantities of BoNT/A, for example, has been used effectively totreat spasticity due to brain injury, spinal cord injury, stroke,multiple sclerosis and cerebral palsy. Additional possible clinical usesof clostridial neurotoxins currently are being investigated.

Given the potential danger associated with small quantities of botulinumtoxins in foodstuffs and the need to prepare accurate pharmaceuticalformulations, assays for botulinum neurotoxins presently are employed inboth the food and pharmaceutical industry. The food industry requiresassays for the botulinum neurotoxins to validate new food packagingmethods and to ensure food safety. The growing clinical use of thebotulinum toxins necessitates accurate assays for botulinum neurotoxinactivity for product formulation as well as quality control. In bothindustries, a mouse lethality test currently is used to assay forbotulinum neurotoxin activity. Unfortunately, this assay suffers fromseveral drawbacks: cost due to the large numbers of laboratory animalsrequired; lack of specificity; and the potential for inaccuracy unlesslarge animal groups are used.

Thus, there is a need for new materials and methods for assaying forclostridial toxin activity. The present invention satisfies this needand provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides clostridial toxin substrates useful inassaying for the protease activity of any clostridial toxin, includingbotulinum toxins of all serotypes as well as tetanus toxins. Aclostridial toxin substrate of the invention contains a donorfluorophore; an acceptor having an absorbance spectrum overlapping theemission spectrum of the donor fluorophore; and a clostridial toxinrecognition sequence that includes a cleavage site, where the cleavagesite intervenes between the donor fluorophore and the acceptor andwhere, under the appropriate conditions, resonance energy transfer isexhibited between the donor fluorophore and the acceptor. Such aclostridial toxin substrate can include, for example, a botulinum toxinrecognition sequence. In one embodiment, a clostridial toxin substrateof the invention includes a botulinum toxin recognition sequence whichis not a botulinum toxin serotype B (BoNT/B) recognition sequence.

The invention also provides a botulinum serotype A/E (BoNT/B) substratecontaining (a) a donor fluorophore; (b) an acceptor having an absorbancespectrum overlapping the emission spectrum of the donor fluorophore; and(c) a BoNT A or BoNT/E recognition sequence containing a cleavage site,where the cleavage site intervenes between the donor fluorophore and theacceptor and where, under the appropriate conditions, resonance energytransfer is exhibited between the donor fluorophore and the acceptor.Such a botulinum serotype NE substrate also can be susceptible tocleavage by both the BoNT/A and BoNT/E toxins.

The invention further provides, for example, a botulinum toxin serotypeA (BoNT/A) substrate containing a donor fluorophore; an acceptor havingan absorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/A recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/A substrate of the invention can include, for example,at least six consecutive residues of SNAP-25, where the six consecutiveresidues include Gln-Arg, or a peptidomimetic thereof. In these andother amino acid sequences provided herein, it is understood that thesequence is written in the direction from N-terminus to C-terminus. ABoNT/A substrate of the invention also can have, for example, at leastsix consecutive residues of human SNAP-25, where the six consecutiveresidues include Gln₁₉₇-Arg₁₉₈, or a peptidomimetic thereof. In oneembodiment, a BoNT/A substrate of the invention includes the amino acidsequence Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO: 1), or apeptidomimetic thereof. In another embodiment, a BoNT/A substrate of theinvention includes residues 187 to 203 of human SNAP-25 (SEQ ID NO: 2),or a peptidomimetic thereof. A variety of donor fluorophores andacceptors are useful in a BoNT/A substrate of the invention, includingbut not limited to, fluorescein-tetramethylrhodamine; DABCYL-EDANS; andALEXA FLUOR®-488-QSY® 7.

Further provided by the invention is a botulinum toxin serotype B(BoNT/B) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/B recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/B substrate of the invention can contain, for example,at least six consecutive residues of VAMP, where the six consecutiveresidues include Gln-Phe, or a peptidomimetic thereof. For example, aBoNT/B substrate of the invention can contain at least six consecutiveresidues of human VAMP-2, the six consecutive residues includingGln₇₆-Phe₇₇, or a peptidomimetic thereof. In one embodiment, a BoNT/Bsubstrate includes the amino acid sequenceGly-Ala-Ser-Gln-Phe-Glu-Thr-Ser (SEQ ID NO: 3), or a peptidomimeticthereof. In another embodiment, a BoNT/B substrate includes residues 55to 94 of human VAMP-2 (SEQ ID NO: 4); residues 60 to 94 of human VAMP-2(SEQ ID NO: 4); or residues 60 to 88 of human VAMP-2 (SEQ ID NO: 4), ora peptidomimetic of one of these sequences. It is understood that avariety of donor fluorophores and acceptors are useful in a BoNT/Bsubstrate of the invention; such donor fluorophore-acceptor combinationsinclude, but are not limited to, fluorescein-tetramethylrhodamine;DABCYL-EDANS; and ALEXA FLUOR®-488-QSY® 7.

The invention also provides a botulinum toxin serotype C1 (BoNT/C1)substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/C1 recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/C1 substrate of the invention can have, for example, atleast six consecutive residues of syntaxin, the six consecutive residuesincluding Lys-Ala, or a peptidomimetic thereof. For example, a BoNT/C1substrate of the invention can have at least six consecutive residues ofhuman syntaxin, the six consecutive residues including Lys₂₅₃-Ala₂₅₄, ora peptidomimetic thereof. In one embodiment, a BoNT/C1 substratecontains the amino acid sequence Asp-Thr-Lys-Lys-Ala-Val-Lys-Tyr (SEQ IDNO: 5), or a peptidomimetic thereof.

A BoNT/C1 substrate of the invention also can contain, for example, atleast six consecutive residues of SNAP-25, where the six consecutiveresidues include Arg-Ala, or a peptidomimetic thereof. Such a BoNT/C1substrate can have, for example, at least six consecutive residues ofhuman SNAP-25, the six consecutive residues including Arg₁₉₈-Ala₁₉₉, ora peptidomimetic thereof. An exemplary BoNT/C1 substrate containsresidues 93 to 202 of human SNAP-25 (SEQ ID NO: 2), or a peptidomimeticthereof. As for all the clostridial toxin substrates of the invention, avariety of donor fluorophore-acceptor combinations are useful in aBoNT/C1 substrate, including, for example,fluorescein-tetramethylrhodamine; DABCYL-EDANS; and ALEXAFLUOR®-488-QSY® 7.

The present invention further provides a botulinum toxin serotype D(BoNT/D) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/D recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/D substrate of the invention can have, for example, atleast six consecutive residues of VAMP, the six consecutive residuesincluding Lys-Leu, or a peptidomimetic thereof. In one embodiment, aBoNT/D substrate contains at least six consecutive residues of humanVAMP, the six consecutive residues including Lys₅₉-Leu₆₀, or apeptidomimetic thereof. In another embodiment, a BoNT/D substrate of theinvention contains the amino acid sequenceArg-Asp-Gln-Lys-Leu-Ser-Glu-Leu (SEQ ID NO: 6), or a peptidomimeticthereof. In a further embodiment, a BoNT/D substrate of the inventionincludes residues 27 to 116 of rat VAMP-2 (SEQ ID NO: 7), or apeptidomimetic thereof. It is understood that a variety of donorfluorophore-acceptor combinations are useful in a BoNT/D substrate ofthe invention; such donor fluorophore-acceptor pairs include, but arenot limited to, fluorescein-tetramethylrhodamine; DABCYL-EDANS; andALEXA FLUOR®-488-QSY® 7.

The present invention additionally provides a botulinum toxin serotype E(BoNT/E) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/E recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/E substrate can contain, for example, at least sixconsecutive residues of SNAP-25, the six consecutive residues includingArg-Ile, or a peptidomimetic thereof. Such a BoNT/E substrate can have,for example, at least six consecutive residues of human SNAP-25, the sixconsecutive residues including Arg₁₈₀-Ile₁₈₁, or a peptidomimeticthereof. In one embodiment, a BoNT/E substrate includes the amino acidsequence Gln-Ile-Asp-Arg-Ile-Met-Glu-Lys (SEQ ID NO: 8), or apeptidomimetic thereof. In another embodiment, a BoNT/E substrateincludes residues 156 to 186 of human SNAP-25 (SEQ ID NO: 2), or apeptidomimetic thereof. A variety of donor fluorophore-acceptorcombinations are useful in a BoNT/E substrate of the invention. Thesedonor fluorophore-acceptor combinations include, without limitation,fluorescein-tetramethylrhodamine; DABCYL-EDANS; and ALEXAFLUOR®-488-QSY® 7.

Further provided by the invention is a botulinum toxin serotype F(BoNT/F) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/F recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. Such a BoNT/F substrate can have, for example, at least sixconsecutive residues of VAMP, the six consecutive residues includingGln-Lys, or a peptidomimetic thereof. In one embodiment, a BoNT/Fsubstrate has at least six consecutive residues of human VAMP, the sixconsecutive residues including Gln₅₈-Lys₅₉, or a peptidomimetic thereof.In another embodiment, a BoNT/F substrate of the invention includesresidues 27 to 116 of rat VAMP-2 (SEQ ID NO: 7), or a peptidomimeticthereof. In a further embodiment, a BoNT/F substrate includes the aminoacid sequence Glu-Arg-Asp-Gln-Lys-Leu-Ser-Glu (SEQ ID NO: 9), or apeptidomimetic thereof. Those skilled in the art of fluorescenceresonance energy transfer understand that a variety of donorfluorophore-acceptor combinations are useful in a BoNT/F substrate ofthe invention, including, as not limiting examples,fluorescein-tetramethylrhodamine; DABCYL-EDANS; and ALEXAFLUOR®-488-QSY® 7.

The present invention also provides a botulinum toxin serotype G(BoNT/G) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/G recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/G substrate can have, for example, at least sixconsecutive residues of VAMP, the six consecutive residues includingAla-Ala, or a peptidomimetic thereof. Such a BoNT/G substrate can have,for example, at least six consecutive residues of human VAMP, the sixconsecutive residues including Ala₈₃-Ala₈₄, or a peptidomimetic thereof.In one embodiment, a BoNT/G substrate contains the amino acid sequenceGlu-Thr-Ser-Ala-Ala-Lys-Leu-Lys (SEQ ID NO: 10), or a peptidomimeticthereof. As discussed above in regard to other clostridial toxinsubstrates, a variety of donor fluorophore-acceptor combinations areuseful in a BoNT/G substrate of the invention. Such donorfluorophore-acceptor combinations include, for example,fluorescein-tetramethylrhodamine; DABCYL-EDANS; and ALEXAFLUOR®-488-QSY® 7.

Also provided by the invention is a tetanus toxin (TeNT) substratecontaining a donor fluorophore; an acceptor having an absorbancespectrum overlapping the emission spectrum of the donor fluorophore; anda TeNT recognition sequence that includes a cleavage site, where thecleavage site intervenes between the donor fluorophore and the acceptorand where, under the appropriate conditions, resonance energy transferis exhibited between the donor fluorophore and the acceptor.

A TeNT substrate of the invention can have, for example, at least sixconsecutive residues of VAMP, the six consecutive residues includeGln-Phe, or a peptidomimetic thereof. For example, such a TeNT substratecan have at least six consecutive residues of human VAMP-2, the sixconsecutive residues including Gln₇₆-Phe₇₇, or a peptidomimetic thereof.In one embodiment, a TeNT substrate contains the amino acid sequenceGly-Ala-Ser-Gln-Phe-Glu-Thr-Ser (SEQ ID NO: 11), or a peptidomimeticthereof. In another embodiment, the TeNT substrate contains residues 33to 94 of human VAMP-2 (SEQ ID NO: 4); residues 25 to 93 of human VAMP-2(SEQ ID NO: 4); or residues 27 to 116 of rat VAMP-2 (SEQ ID NO: 7), or apeptidomimetic of one of these sequences. A variety of donorfluorophore-acceptor combinations are useful in a TeNT substrate of theinvention, including, without limitation,fluorescein-tetramethylrhodamine; DABCYL-EDANS; and ALEXAFLUOR®-488-QSY® 7.

In specific embodiments, the invention provides a BoNT/A, BoNT/B,BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT substrate that iscleaved with an activity of at least 1 nanomoles/minute/milligram toxin.In other embodiments, a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F,BoNT/G or TeNT substrate of the invention is cleaved with an activity ofat least 10 nanomoles/minute/milligram toxin. In further embodiments, aBoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNTsubstrate of the invention is cleaved with an activity of at least 20nanomoles/minute/milligram toxin. In yet other embodiments, a BoNT/A,BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT substrate of theinvention is cleaved with an activity of at least 50, 100 or 150nanomoles/minute/milligram toxin.

A variety of donor fluorophores and acceptors, including fluorescent andnon-fluorescent acceptors, are useful in the clostridial toxinsubstrates of the invention. Donor fluorophores useful in the inventioninclude, but are not limited to, fluorescein, ALEXA FLUOR®-488, DABCYL,and BODIPY®. Acceptors useful in the invention include, but are notlimited to, tetramethylrhodamine, EDANS and QSY® 7. Exemplary donorfluorophore-acceptor pairs useful in a clostridial toxin substrate ofthe invention include, without limitation,fluorescein-tetramethylrhodamine, ALEXA FLUOR®-488-tetramethylrhodamine,DABCYL-EDANS, fluorescein-QSY® 7, and ALEXA FLUOR®-488-QSY® 7.

Clostridial toxin substrates of the invention encompass peptides andpeptidomimetics of a variety of lengths and in which the donorfluorophore and acceptor are separated by different numbers of residues.In particular embodiments, a clostridial toxin substrate of theinvention is a peptide or peptidomimetic having at most 20 residues, atmost 40 residues, at most 50 residues, or at most 100 residues. In otherembodiments, the donor fluorophore and the acceptor are separated by atmost six residues, at most eight residues, at most ten residues or atmost fifteen residues.

Further provided by the invention is a method of determining clostridialtoxin protease activity. The method includes the steps of (a) treating asample, under conditions suitable for clostridial toxin proteaseactivity, with a clostridial toxin substrate that contains a donorfluorophore, an acceptor having an absorbance spectrum overlapping theemission spectrum of the donor fluorophore, and a clostridial toxinrecognition sequence containing a cleavage site, where the cleavage siteintervenes between the donor fluorophore and the acceptor and where,under the appropriate conditions, resonance energy transfer is exhibitedbetween the donor fluorophore and the acceptor; (b) exciting the donorfluorophore; and (c) determining resonance energy transfer of thetreated substrate relative to a control substrate, where a difference inresonance energy transfer of the treated substrate as compared to thecontrol substrate is indicative of clostridial toxin protease activity.A method of the invention can be practiced with a fluorescent ornon-fluorescent acceptor.

A method of the invention can be used to assay the protease activity ofany clostridial toxin. In one embodiment, a method of the inventionrelies on a BoNT/A substrate to determine BoNT/A protease activity. ABoNT/A substrate useful in a method of the invention can be any of theBoNT/A substrates disclosed herein, for example, a BoNT/A substratecontaining at least six consecutive residues of SNAP-25, where the sixconsecutive residues include Gln-Arg. In another embodiment, a method ofthe invention relies on a BoNT/B substrate to determine BoNT/B proteaseactivity. A BoNT/B substrate useful in a method of the invention can beany of the BoNT/B substrates disclosed herein, for example, a BoNT/Bsubstrate containing at least six consecutive residues of VAMP, wherethe six consecutive residues include Gln-Phe. A method of the inventionalso can utilize a BoNT/C1 substrate to determine BoNT/C1 proteaseactivity. A BoNT/C1 substrate useful in a method of the invention can beany of the BoNT/C1 substrates disclosed herein, for example, a BoNT/C1substrate containing at least six consecutive residues of syntaxin,where the six consecutive residues include Lys-Ala, or containing atleast six consecutive residues of SNAP-25, where the six consecutiveresidues include Arg-Ala.

In another embodiment, a method of the invention relies on a BoNT/Dsubstrate to determine BoNT/D protease activity. A BoNT/D substrateuseful in a method of the invention can be any of the BoNT/D substratesdisclosed herein, for example, a BoNT/D substrate containing at leastsix consecutive residues of VAMP, where the six consecutive residuesinclude Lys-Leu. In a further embodiment, a method of the inventionrelies on a BoNT/E substrate to determine BoNT/E protease activity. ABoNT/E substrate useful in a method of the invention can be any of theBoNT/E substrates disclosed herein, for example, a BoNT/E substratecontaining at least six consecutive residues of SNAP-25, where the sixconsecutive residues include Arg-Ile. In yet a further embodiment, amethod of the invention relies on a BoNT/F substrate to determine BoNT/Fprotease activity. A BoNT/F substrate useful in a method of theinvention can be any of the BoNT/F substrates disclosed herein, forexample, a BoNT/F substrate containing at least six consecutive residuesof VAMP, where the six consecutive residues include Gln-Lys.

A method of the invention also can utilize a BoNT/G substrate todetermine BoNT/G protease activity. A BoNT/G substrate useful in amethod of the invention can be any of the BoNT/G substrates disclosedherein, for example, a BoNT/G substrate containing at least sixconsecutive residues of VAMP, where the six consecutive residues includeAla-Ala. A method of the invention also can be useful to determine TeNTprotease activity and, in this case, relies on a TeNT substrate. Any ofthe TeNT substrates disclosed herein can be useful in a method of theinvention, for example, a TeNT substrate containing at least sixconsecutive residues of VAMP, where the six consecutive residues includeGln-Phe.

A variety of samples that potentially contain an active clostridialtoxin, or light chain or fragment thereof, are useful in the methods ofthe invention. Such samples include, but are not limited to, crude celllysates; isolated clostridial toxins; isolated clostridial toxin lightchains; formulated clostridial toxin products, for example, BOTOX®; andfoodstuffs, including raw, cooked, partially cooked or processed foodsor beverages.

In a method of the invention, resonance energy transfer can bedetermined by a variety of means. In one embodiment, the step ofdetermining resonance energy transfer includes detecting donorfluorescence intensity of the treated substrate, where increased donorfluorescence intensity of the treated substrate as compared to thecontrol substrate is indicative of clostridial toxin protease activity.In another embodiment, the step of determining resonance energy transferincludes detecting acceptor fluorescence intensity of the treatedsubstrate, where decreased acceptor fluorescence intensity of thetreated substrate as compared to the control substrate is indicative ofclostridial toxin protease activity. In a further embodiment, the stepof determining resonance energy transfer includes detecting an acceptoremission maximum and a donor fluorophore emission maximum of the treatedsubstrate, where a shift in emission maxima from near the acceptoremission maximum to near the donor fluorophore emission maximum isindicative of clostridial toxin protease activity. In an additionalembodiment, the step of determining resonance energy transfer includesdetecting the ratio of fluorescence amplitudes near an acceptor emissionmaximum to the fluorescence amplitudes near a donor fluorophore emissionmaximum, where a decreased ratio of the treated sample as compared to acontrol sample is indicative of clostridial toxin protease activity. Inyet a further embodiment, the step of determining resonance energytransfer is practiced by detecting the excited state lifetime of thedonor fluorophore, where an increased donor fluorophore excited statelifetime of the treated substrate as compared to the control substrateis indicative of clostridial toxin protease activity.

As discussed further below, a variety of conditions suitable forclostridial toxin protease activity are useful in a method of theinvention. In one embodiment, the conditions suitable for clostridialtoxin protease activity are selected such that the assay is linear. Inanother embodiment, conditions suitable for clostridial toxin proteaseactivity are selected such that at least 90% of the clostridial toxinsubstrate is cleaved. In a further embodiments, conditions suitable forclostridial toxin protease activity are selected such that at most 5%,at most 10%, at most 15%, at most 20% or at most 25% of the clostridialtoxin substrate is cleaved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the deduced structure and postulatedmechanism of activation of clostridial neurotoxins. Toxins can beproduced as an inactive single polypeptide chain of 150 kDa, composed ofthree 50 kDa domains connected by loops. Selective proteolytic cleavageactivates the toxins by generating two disulfide-linked chains: the Lchain of 50 kDa and the H chain of 100 kDa, which is made up of twodomains denoted H_(N) and H_(C). The three domains play distinct roles:the C-terminal domain of the heavy chain (H_(C)) functions in cellbinding while the N-terminal domain of the heavy chain (H_(N)) permitstranslocation from endosome to cell cytoplasm. Following reduction ofthe disulfide linkage inside the cell, the zinc-endopeptidase activityof the L chain is liberated.

FIG. 2 shows a schematic of the four steps required for tetanus andbotulinum toxin activity in central and peripheral neurons.

FIG. 3 shows the subcellular localization at the plasma membrane andsites of cleavage of SNAP-25, VAMP and syntaxin. VAMP is bound tosynaptic vesicle membrane, whereas SNAP-25 and syntaxin are bound to thetarget plasma membrane. BoNT/A and /E cleave SNAP-25 close to thecarboxy-terminus, releasing nine or 26 residues, respectively. BoNT/B,/D, /F, /G and TeNT act on the conserved central portion of VAMP(dotted) and release the amino-terminal portion of VAMP into thecytosol. BoNT/C1 cleaves SNAP-25 close to the carboxy-terminus as wellas cleaving syntaxin at a single site near the cytosolic membranesurface. The action of BoNT/B, /C1, /D, /F, /G and TeNT results inrelease of a large portion of the cytosolic domain of VAMP or syntaxin,while only a small portion of SNAP-25 is released by selectiveproteolysis by BoNT/A, /C1 or /E.

FIG. 4 shows the neurotoxin recognition motif of VAMP, SNAP-25 andsyntaxin. (A) Hatched boxes indicate the presence and positions of amotif common to the three targets of clostridial neurotoxins. (B) Therecognition motif is composed of hydrophobic residues (“h”); negativelycharged Asp or Glu residues (“−”) and polar residues (“p”); “x”represents any amino acid. The motif is included in regions of VAMP,SNAP-25 and syntaxin predicted to adopt an α-helical conformation. (C) Atop view of the motif in an α-helical conformation is shown. Negativelycharged residues align on one face, while hydrophobic residues align ona second face.

FIG. 5 shows an alignment of various SNAP-25 proteins and their BoNT/E,BoNT/A and BoNT/C1 cleavage sites. Human SNAP-25 (SEQ ID NO: 2; GenBankaccession g4507099; see, also, related human SNAP-25 sequence g2135800);mouse SNAP-25 (SEQ ID NO: 12; GenBank accession G6755588); DrosophilaSNAP-25 (SEQ ID NO: 13; GenBank accession g548941); goldfish SNAP-25(SEQ ID NO: 14; GenBank accession g2133923); sea urchin SNAP-25 (SEQ IDNO: 15; GenBank accession g2707818) and chicken SNAP-25 (SEQ ID NO: 16;GenBank accession g481202) are depicted.

FIG. 6 shows an alignment of various VAMP proteins and their BoNT/F,BoNT/D, BoNT/B, TeNT and BoNT/G cleavage sites. Human VAMP-1 (SEQ ID NO:96; GenBank accession g135093); human VAMP-2 (SEQ ID NO: 4; GenBankaccession g135094); mouse VAMP-2 (SEQ ID NO: 17; GenBank accessiong2501081); bovine VAMP (SEQ ID NO: 15; GenBank accession g89782); frogVAMP (SEQ ID NO: 19; GenBank accession g6094391); and sea urchin VAMP(SEQ ID NO: 20; GenBank accession g5031415) are depicted.

FIG. 7 shows an alignment of various syntaxin proteins and their BoNT/C1cleavage sites. Human syntaxin 1A (SEQ ID NO: 21; GenBank accessiong15079184), human syntaxin 1B2 (SEQ ID NO: 22; GenBank accessiong15072437), mouse syntaxin 1A (SEQ ID NO: 23; GenBank accessiong15011853), Drosophila syntaxin 1A (SEQ ID NO: 24; GenBank accessiong2501095); C. elegans syntaxin A (SEQ ID NO: 25; GenBank accessiong7511662) and sea urchin syntaxin (SEQ ID NO: 26; GenBank accessiong13310402) are depicted.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides clostridial toxin substrates useful indetermining the presence or absence of a clostridial toxin or fordetermining the protease activity of any clostridial toxin, includingbotulinum toxins of all serotypes as well as tetanus toxins. Theclostridial toxin substrates of the invention are valuable, in part,because they can be utilized in rapid and simple homogeneous screeningassays that do not require separation of cleaved product from uncleavedsubstrate and do not rely on toxicity to animals. Furthermore, theclostridial toxin substrates of the invention can be used, for example,to analyze crude and bulk samples as well as highly purified dichaintoxins or isolated clostridial toxin light chains.

As discussed below, fluorescence resonance energy transfer (FRET) is adistance-dependent interaction between the electronic excited states oftwo molecules in which excitation is transferred from a donorfluorophore to an acceptor without emission of a photon. The process ofenergy transfer results in a reduction (quenching) of fluorescenceintensity and excited state lifetime of the donor fluorophore and, wherethe acceptor is a fluorophore, can produce an increase in the emissionintensity of the acceptor. Upon cleavage of a clostridial toxinsubstrate of the invention, resonance energy transfer is reduced and canbe detected, for example, by increased donor fluorescence emission,decreased acceptor fluorescence emission, or by a shift in the emissionmaxima from near the acceptor emission maxima to near the donor emissionmaxima. If desired, the amount of clostridial toxin in a sample can becalculated as a function of the difference in the degree of FRET usingthe appropriate standards.

A clostridial toxin substrate of the invention contains a donorfluorophore; an acceptor having an absorbance spectrum overlapping theemission spectrum of the donor fluorophore; and a clostridial toxinrecognition sequence that includes a cleavage site, where the cleavagesite intervenes between the donor fluorophore and the acceptor andwhere, under the appropriate conditions, resonance energy transfer isexhibited between the donor fluorophore and the acceptor. A clostridialtoxin substrate of the invention can include, for example, a botulinumtoxin recognition sequence. In one embodiment, a clostridial toxinsubstrate of the invention includes a botulinum toxin recognitionsequence which is not a botulinum toxin serotype B (BoNT/B) recognitionsequence.

In specific embodiments, the invention provides a BoNT/A, BoNT/B,BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT substrate that iscleaved with an activity of at least 1 nanomole/minute/milligram toxin.In other embodiments, a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F,BoNT/G or TeNT substrate of the invention is cleaved with an activity ofat least 10 nanomoles/minute/milligram toxin. In further embodiments, aBoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNTsubstrate of the invention is cleaved with an activity of at least 20nanomoles/minute/milligram toxin. In yet other embodiments, a BoNT/A,BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT substrate of theinvention is cleaved with an activity of at least 50, 100 or 150nanomoles/minute/milligram toxin. It is understood that such activity ismeasured under standard kinetic conditions.

A variety of donor fluorophores and acceptors, including fluorescent andnon-fluorescent acceptors, are useful in the clostridial toxinsubstrates of the invention. Donor fluorophores useful in the inventioninclude, but are not limited to, fluorescein, ALEXA FLUOR®-488, DABCYL,and BODIPY®. Acceptors useful in the invention include, but are notlimited to, tetramethylrhodamine, EDANS and QSY® 7. Exemplary donorfluorophore-acceptor pairs useful in a clostridial toxin substrate ofthe invention include, without limitation,fluorescein-tetramethylrhodamine, ALEXA FLUOR®-488-tetramethylrhodamine,DABCYL-EDANS, fluorescein-QSY® 7, and ALEXA FLUOR®-488-QSY® 7.

Clostridial toxin substrates of the invention encompass proteins,peptides and peptidomimetics of a variety of lengths and in which thedonor fluorophore and acceptor are separated by different numbers ofresidues. In particular embodiments, a clostridial toxin substrate ofthe invention is has at most 20 residues, at most 40 residues, at most50 residues, at most 100 residues, at most 150 residues, at most 200residues, at most 250 residues, at most 300 residues, at most 350residues or at most 400 residues. In other embodiments, the donorfluorophore and the acceptor are separated by at most six residues, atmost eight residues, at most ten residues, at most twelve residues, atmost fifteen residues, at most twenty residues, at most twenty-fiveresidues, at most thirty residues, at most thirty-five residues or atmost forty residues.

Tetanus and botulinum neurotoxins are produced by Clostridia and causethe neuroparalytic syndromes of tetanus and botulism. While tetanusneurotoxin acts mainly at the CNS synapse, botulinum neurotoxins actperipherally. Clostridial neurotoxins share a similar mechanism of cellintoxication, blocking the release of neurotransmitters. In thesetoxins, which are composed of two disulfide-linked polypeptide chains,the larger subunit is responsible for neurospecific binding andtranslocation of the smaller subunit into the cytoplasm. Upontranslocation and reduction in neurons, the smaller chain displayspeptidase activity specific for protein components involved inneuroexocytosis in the neuronal cytosol. The SNARE protein targets ofclostridial toxins are common to exocytosis in a variety of non-neuronaltypes; in these cells, as in neurons, light chain peptidase activityinhibits exocytosis.

Tetanus neurotoxin and botulinum neurotoxins B, D, F, and G recognizespecifically VAMP (synaptobrevin), an integral protein of the synapticvesicle membrane which is cleaved at distinct bonds depending on theneurotoxin. Botulinum A and E neurotoxins recognize and cleavespecifically SNAP-25, a protein of the presynaptic membrane, at twodifferent sites in the carboxy-terminal portion of the protein.Botulinum neurotoxin C cleaves syntaxin, a protein of the nerveplasmalemma, in addition to SNAP-25. The three protein targets of theClostridial neurotoxins are conserved from yeast to humans althoughcleavage sites and toxin susceptibility are not necessarily conserved(see below; see, also, Humeau et al., Biochimie 82:427-446 (2000);Niemann et al., Trends in Cell Biol. 4:179-185 (1994); and Pellizzari etal., Phil. Trans. R. Soc. London 354:259-268 (1999)).

Naturally occurring tetanus and botulinum neurotoxins are produced asinactive polypeptide chains of 150 kDa without a leader sequence. Thesetoxins may be cleaved by bacterial or tissue proteinases at an exposedprotease-sensitive loop, generating active di-chain toxin. Naturallyoccurring clostridial toxins contain a single interchain disulfide bondbridging the heavy chain (H, 100 kDa) and light chain (L, 50 kDa); sucha bridge is important for neurotoxicity of toxin added extracellularly(Montecucco and Schiavo, Quarterly Rev. Biophysics 28:423-472 (1995)).

The clostridial toxins appear to be folded into three distinct 50 kDadomains, as shown in FIG. 1, with each domain having a distinctfunctional role. AS illustrated in FIG. 2, the cell intoxicationmechanism of the clostridial toxins consists of four distinct steps: (1)binding; (2) internalization; (3) membrane translocation; and (4)enzymatic target modification. The carboxy-terminal part of the heavychain (H_(C)) functions in neurospecific binding, while theamino-terminal portion of the H chain (H_(N)) functions in membranetranslocation. The L chain is responsible for the intracellularcatalytic activity (Montecucco and Schiavo, supra, 1995).

The amino acid sequence of eight human clostridial neurotoxins has beenderived from the corresponding gene (Neimann, “Molecular Biology ofClostridial Neurotoxins” in Sourcebook of Bacterial Protein Toxins Aloufand Freer (Eds.) pp. 303-348 London: Academic Press 1991). The L chainsand H chains are composed of roughly 439 and 843 residues, respectively.Homologous segments are separated by regions of little or no similarity.The most well conserved regions of the L chains are the amino-terminalportion (100 residues) and central region (corresponding to residues 216to 244 of TeNT), as well as the two cysteines forming the interchaindisulfide bond. The 216 to 244 region contains a His-Glu-X-X-His bindingmotif characteristic of zinc-endopeptidases.

The heavy chains are less well conserved than the light chains, and thecarboxy-terminal part of H_(C) (corresponding to residues 1140 to 1315of TeNT) is the most variable. This is consistent with the involvementof the H_(C) domain in binding to nerve terminals and the fact that thedifferent neurotoxins appear to bind different receptors. Notsurprisingly, many serotype specific antibodies recognize heavy chaindeterminants.

Comparison of the nucleotide and amino acid sequences of clostridialtoxins indicates that they derive from a common ancestral gene.Spreading of these genes may have been facilitated by the fact that theclostridial neurotoxin genes are located on mobile genetic elements. Asdiscussed further below, sequence variants of the seven botulinum toxinsare known in the art. See, for example, FIGS. 5 to 7 and Humeau et al.,supra, 2000.

As discussed above, natural targets of the clostridial neurotoxinsinclude VAMP, SNAP-25, and syntaxin. VAMP is bound to the synapticvesicle membrane, whereas SNAP-25 and syntaxin are bound to the targetmembrane (see FIG. 3). BoNT/A and BoNT/E cleave SNAP-25 in thecarboxy-terminal region, releasing nine or twenty-six amino acidresidues, respectively, and BoNT/C1 also cleaves SNAP-25 near thecarboxy-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F andBoNT/G, and tetanus toxin, act on the conserved central portion of VAMP,and release the amino-terminal portion of VAMP into the cytosol. BoNT/C1cleaves syntaxin at a single site near the cytosolic membrane surface.Thus, the action of BoNT/B, BoNT/C1, BoNT/D, BoNT/F, BoNT/G and TeNTresults in release of a large portion of the cytosolic domain of VAMPand syntaxin, while only a small portion of SNAP-25 is released byproteolysis of BoNT/A, BoNT/C1 or BoNT/E (Montecucco and Schiavo, supra,1995).

SNAP-25, a protein of about 206 residues lacking a transmembranesegment, is associated with the cytosolic surface of the nerveplasmalemma (FIG. 3; see, also, Hodel et al., Int. J. Biochemistry andCell Biology 30:1069-1073 (1998)). In addition to homologs highlyconserved from Drosophila to mammals, SNAP-25-related proteins also havebeen cloned from yeast. SNAP-25 is required for axonal growth duringdevelopment and may be required for nerve terminal plasticity in themature nervous system. In humans, two isoforms are differentiallyexpressed during development; isoform a is constitutively expressedbeginning in the embryo stage, while isoform b appears at birth andpredominates in adult life. SNAP-25 analogues such as SNAP-23 also areexpressed outside the nervous system, for example, in pancreatic cells.

VAMP is a protein of about 120 residues, with the exact length dependingon the species and isotype. As shown in FIG. 3, VAMP contains a shortcarboxy-terminal segment inside the vesicle lumen while most of themolecule is exposed to the cytosol. The proline-rich amino-terminalthirty residues are divergent among species and isoforms while thecentral portion of VAMP (residues 30 to 96), which is rich in chargedand hydrophilic residues and includes known cleavage sites, is highlyconserved. VAMP is associated on the synaptic vesicle membrane withsynaptophysin.

A variety of species homologs of VAMP are known in the art includinghuman, rat, bovine, Torpedo, Drosophila, yeast, squid and Aplysiahomologs. In addition, multiple isoforms of VAMP have been identifiedincluding VAMP-1, VAMP-2 and cellubrevin, and insensitive forms havebeen identified in non-neuronal cells. VAMP appears to be present in allvertebrate tissues although the distribution of VAMP-1 and VAMP-2 variesin different cell types. Chicken and rat VAMP-1 are not cleaved by TeNTor BoNT/B. These VAMP-1 homologs have a valine in place of glutaminepresent in human and mouse VAMP-1 at the TeNT or BoNT/B cleavage site.The substitution does not effect BoNT/D, /F or /G, which cleave bothVAMP-1 and VAMP-2 with similar rates.

Syntaxin, located on the cytosolic surface of the nerve plasmalemma, ismembrane-anchored via a carboxy-terminal segment with most of theprotein exposed to the cytosol. Syntaxin colocalizes with calciumchannels at the active zones of the presynaptic membrane, whereneurotransmitter release takes place. In addition, syntaxin interactswith synaptotagmin, a protein of the SSV membrane, that forms afunctional bridge between the plasmalemma and the vesicles. A variety ofsyntaxin isoforms have been identified. Two isoforms of slightlydifferent length (285 and 288 residues) have been identified in nervecells (isoforms 1A and 1B), with isoforms 2, 3, 4 and 5 present in othertissues. The isoforms have varying sensitivities to BoNT/C1, with the1A, 1B, 2 and 3 syntaxin isoforms cleaved by this toxin.

A clostridial toxin substrate of the invention contains a donorfluorophore; an acceptor having an absorbance spectrum overlapping theemission spectrum of the donor fluorophore; and a clostridial toxinrecognition sequence that includes a cleavage site, where the cleavagesite intervenes between the donor fluorophore and the acceptor andwhere, under the appropriate conditions, resonance energy transfer isexhibited between the donor fluorophore and the acceptor. Thus, aclostridial toxin substrate is a polypeptide, peptide or peptidomimeticthat is susceptible to cleavage by at least one clostridial toxin underconditions suitable for clostridial toxin protease activity.

As used herein, the term “donor fluorophore” means a molecule that, whenirradiated with light of a certain wavelength, emits light, also denotedfluorescence, of a different wavelength. The term fluorophore issynonymous in the art with the term “fluorochrome.”

The term “acceptor,” as used herein, refers to a molecule that canabsorb energy from, and upon excitation of, a donor fluorophore and is aterm that encompasses fluorophores as well as non-fluorescent molecules.An acceptor useful in a clostridial toxin substrate of the invention hasan absorbance spectrum which overlaps the emission spectrum of a donorfluorophore. An acceptor useful in the invention generally also hasrather low absorption at a wavelength suitable for excitation of thedonor fluorophore.

In a clostridial toxin substrate of the invention, an acceptor has anabsorbance spectrum that overlaps the emission spectrum of the donorfluorophore. The term “overlapping,” as used herein in reference to theabsorbance spectrum of an acceptor and the emission spectrum of a donorfluorophore, means an absorbance spectrum and emission spectrum that arepartly or entirely shared. Thus, in such overlapping spectra, the highend of the range of the donor fluorophore's emission spectrum is higherthan the low end of the range of the acceptor's absorbance spectrum.

As used herein, the term “clostridial toxin recognition sequence” meansa scissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aclostridial toxin under conditions suitable for clostridial toxinprotease activity.

A clostridial toxin substrate of the invention contains a cleavage sitethat “intervenes” between a donor fluorophore and an acceptor having anabsorbance spectrum which overlaps the emission spectrum of the donorfluorophore. Thus, the cleavage site is positioned in between thefluorophore and acceptor such that cleavage at the site results in afirst molecule containing the fluorophore and a second moleculecontaining the acceptor. It is understood that all or only a portion ofthe clostridial toxin recognition sequence can intervene between thedonor fluorophore and acceptor.

The invention further provides a “composite” clostridial toxinsubstrate. Such a composite clostridial toxin substrate contains (a) afirst member of a donor fluorophore-acceptor pair linked to a firstpartner of an affinity couple; and (b) a clostridial toxin recognitionsequence containing a cleavage site, where the recognition sequence islinked to a second member of the donor fluorophore-acceptor pair and asecond partner of the affinity couple, where the cleavage siteintervenes between the second member of the donor fluorophore-acceptorpair and the second partner of the affinity couple, and where (a) and(b) are stably associated such that, under the appropriate conditions,resonance energy transfer is exhibited between the first and secondmembers of the donor fluorophore-acceptor pair. Thus, a compositeclostridial toxin substrate of the invention is, in effect, a bipartiteclostridial toxin substrate in which the two parts are stably associatedthrough the affinity couple. As for other clostridial toxin substrates,resonance energy transfer is altered upon cleavage of the compositesubstrate. It is understood that the clostridial toxin recognitionsequences and cleavage sites described herein and well known in the artcan be useful in composite clostridial toxin substrates as well as innon-composite clostridial toxin substrates, which do not necessarilycontain an affinity couple.

The term “donor fluorophore-acceptor pair,” as used herein, means adonor fluorophore and an acceptor that has an absorbance spectrumoverlapping the emission spectrum of the donor fluorophore. Where thefirst member of the pair is a donor fluorophore, the second member ofthe pair will be an acceptor. Where the first member of the pair is anacceptor, the second member of the pair will be a donor fluorophore.

In one embodiment, the first member of the donor fluorophore-acceptorpair is a donor fluorophore, and the second member is an acceptor. Inanother embodiment, the first member of the donor fluorophore-acceptorpair is an acceptor, and the second member is a donor fluorophore. Avariety of donor fluorophores and acceptors are useful in the compositeclostridial toxin substrates of the invention, including the donorfluorophores and acceptors described herein. In one embodiment, thedonor fluorophore is a lanthanide. Lanthanide donor fluorophores usefulin a composite substrate of the invention include, without limitation,terbium, europium, dysprosium and samarium.

The term “affinity couple,” as used herein, means two molecules that arecapable of forming a stable, non-covalent association. Affinity couplesuseful in a composite substrate of the invention include, withoutlimitation, streptavidin-biotin; S peptide-S protein; histidinetag-nickel chelate; antibody-antigen, for example, FLAG and anti-FLAGantibody; and receptor-ligand.

In one embodiment, the affinity couple is streptavidin-biotin. In afurther embodiment, the first partner of the affinity couple isstreptavidin, and the second partner is biotin. In another embodiment,the first partner of the affinity couple is biotin, and the secondpartner is streptavidin. In yet further embodiments, the affinity coupleis streptavidin-biotin, and the donor fluorophore is terbium, europium,dysprosium or samarium.

Clostridial toxins have specific and distinct cleavage sites. BoNT/Acleaves a Gln-Arg bond; BoNT/B and TeNT cleaves a Gln-Phe bond; BoNT/C1cleaves a Lys-Ala or Arg-Ala bond; BoNT/D cleaves a Lys-Leu bond; BoNT/Ecleaves an Arg-Ile bond; BoNT/F cleaves a Gln-Lys bond; and BoNT/Gcleaves an Ala-Ala bond (see Table 1). The scissile bond can berepresented P₁-P₁′, and it is understood that a P₁ or P₁′ site, or both,can be substituted with another amino acid or amino acid mimetic inplace of the naturally occurring residue. For example, BoNT/A substrateshave been prepared in which the P₁ position (Gln) is modified to be analanine, 2-aminobutyric acid or asparagine residue; these substrateswere hydrolyzed by BoNT/A at the P₁-Arg bond (Schmidt and Bostian, J.Protein Chem. 16:19-26 (1997)). However, it is recognized thatsubstitutions can be introduced at the P₁ position of the scissile bond,for example, a BoNT/A scissile bond, while conservation of the P₁′residue is more often important for detectable proteolysis (Vaidyanathanet al., J. Neurochem. 72:327-337 (1999)). Thus, in one embodiment, theinvention provides a clostridial toxin substrate in which the P₁′residue is not modified or substituted relative to the naturallyoccurring residue in a target protein cleaved by the clostridial toxin.In another embodiment, the invention provides a clostridial toxinsubstrate in which the P₁ residue is modified or substituted relative tothe naturally occurring residue in a target protein cleaved by theclostridial toxin; such a substrate retains susceptibility to peptidebond cleavage between the P₁ and P₁′ residues.

TABLE 1 Bond cleaved in human VAMP-2, SNAP-25 or syntaxin Toxin TargetP₄-P₃-P₂-P₁ -- P₁′-P₂′-P₃′-P₄′ SEQ ID NO BoNT/A SNAP-25Glu-Ala-Asn-Gln-Arg*-Ala-Thr-Lys SEQ ID NO: 1 BoNT/B VAMP-2Gly-Ala-Ser-Gln-Phe*-Glu-Thr-Ser SEQ ID NO: 3 BoNT/C1 syntaxinAsp-Thr-Lys-Lys-Ala*-Val-Lys-Tyr SEQ ID NO: 5 BoNT/D VAMP-2Arg-Asp-Gln-Lys-Leu*-Ser-Glu-Leu SEQ ID NO: 6 BoNT/E SNAP-25Gln-Ile-Asp-Arg-Ile*-Met-Glu-Lys SEQ ID NO: 8 BoNT/F VAMP-2Glu-Arg-Asp-Gln-Lys*-Leu-Ser-Glu SEQ ID NO: 9 BoNT/G VAMP-2Glu-Thr-Ser-Ala-Ala*-Lys-Leu-Lys SEQ ID NO: 10 TeNT VAMP-2Gly-Ala-Ser-Gln-Phe*-Glu-Thr-Ser SEQ ID NO: 11 *Scissile bond shown inbold

SNAP-25, VAMP and syntaxin share a short motif located within regionspredicted to adopt an α-helical conformation (see FIG. 4). This motif ispresent in SNAP-25, VAMP and syntaxin isoforms expressed in animalssensitive to the neurotoxins. In contrast, Drosophila and yeast homologsthat are resistant to these neurotoxins and syntaxin isoforms notinvolved in exocytosis contain sequence variations in the α-helicalmotif regions of these VAMP and syntaxin proteins.

Multiple repetitions of the α-helical motif are present in proteinssensitive to cleavage by clostridial toxins: four copies are naturallypresent in SNAP-25; two copies are naturally present in VAMP; and twocopies are naturally present in syntaxin (see FIG. 4A). Furthermore,peptides corresponding to the specific sequence of the α-helical motifscan inhibit neurotoxin activity in vitro and in vivo, and such peptidescan cross-inhibit different neurotoxins. In addition, antibodies raisedagainst such peptides can cross-react among the three target proteins,indicating that this α-helical motif is exposed on the cell surface andadopts a similar configuration in each of the three target proteins.Consistent with these findings, SNAP-25-specific, VAMP-specific andsyntaxin-specific neurotoxins cross-inhibit each other by competing forthe same binding site, although they do not cleave targetsnon-specifically. These results indicate that a clostridial toxinrecognition sequence can include, if desired, at least one α-helicalmotif. It is recognized that an α-helical motif is not absolutelyrequired for cleavage by a clostridial toxin as evidenced by 16-mer and17-mer substrates for BoNT/A, as discussed further below.

Although multiple α-helical motifs are found in SNAP-25, VAMP andsyntaxin, in one embodiment the invention provides a clostridial toxinsubstrate in which the clostridial toxin recognition sequence includes asingle α-helical motif. In another embodiment, the invention provides aclostridial toxin substrate in which the clostridial toxin recognitionsequence includes two or more α-helical motifs. A BoNT/A or BoNT/Erecognition sequence can include, for example, the S4 α-helical motif,alone or combined with one or more additional α-helical motifs; BoNT/B,BoNT/G or TeNT recognition sequence can include, for example, the V2α-helical motif, alone or combined with one or more additional α-helicalmotifs; a BoNT/C1 recognition sequence can include, for example, the S4α-helical motif, alone or combined with one or more additional α-helicalmotifs, or X2 α-helical motif, alone or combined with one or moreadditional α-helical motifs; and a BoNT/D or BoNT/F recognition sequencecan include, for example, the V1 α-helical motif, alone or combined withone or more additional α-helical motifs (see FIG. 4A).

A clostridial toxin substrate of the invention can contain one ormultiple clostridial toxin cleavage sites for the same or differentclostridial toxin. In one embodiment, a clostridial toxin substrate ofthe invention contains a single cleavage site. In another embodiment, aclostridial toxin substrate of the invention has multiple cleavage sitesfor the same clostridial toxin. These cleavage sites can be accompaniedby the same or different clostridial toxin recognition sequences. In afurther embodiment, a clostridial toxin substrate of the invention hasmultiple cleavage sites for the same clostridial toxin that intervenebetween the same donor fluorophore and acceptor. A clostridial toxinsubstrate of the invention can contain, for example, two or more, threeor more, five or more, or ten or more cleavage sites for the sameclostridial toxin intervening between the same or different donorfluorophore-acceptor pairs. A clostridial substrate of the inventionalso can have, for example, two, three, four, five, six, seven, eight,nine or ten cleavage sites for the same clostridial toxin interveningbetween the same or different donor fluorophore-acceptor pairs.

A clostridial toxin substrate of the invention containing multiplecleavage sites can contain cleavage sites and recognition sequences fordifferent clostridial toxins. In one embodiment, a clostridial toxinsubstrate of the invention includes multiple cleavage sites fordifferent clostridial toxins all intervening between the same donorfluorophore-acceptor pair. A clostridial toxin substrate of theinvention can contain, for example, two or more, three or more, five ormore, or ten or more cleavage sites for different clostridial toxins allintervening between the same donor fluorophore-acceptor pair. Aclostridial toxin substrate of the invention also can contain, forexample, two or more, three or more, five or more, or ten or morecleavage sites for different clostridial toxins intervening between atleast two donor fluorophore-acceptor pairs. In particular embodiments, aclostridial substrate of the invention also has two, three, four, five,six, seven, eight, nine or ten cleavage sites for different clostridialtoxins, where the cleavage sites intervene between the same or differentdonor fluorophore-acceptor pairs. A clostridial toxin substrate of theinvention having multiple cleavage sites can have, for example, anycombination of two, three, four, five, six, seven or eight cleavagesites for any combination of the following clostridial toxins: BoNT/A,BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT.

It is understood that a clostridial toxin substrate of the invention canbe cleaved at a reduced or enhanced rate relative to SNAP-25, VAMP orsyntaxin or relative to a similar peptide or peptidomimetic that doesnot contain extrinsic fluorophores. A clostridial toxin substrate of theinvention such as a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F,BoNT/G or TeNT substrate, can be cleaved, for example, with an initialhydrolysis rate that is at least 5% of the initial hydrolysis rate,under otherwise identical conditions, of human SNAP-25, VAMP orsyntaxin, where the clostridial toxin substrate and SNAP-25, VAMP orsyntaxin each is present at a concentration of 1.0 mM.

Thus, a BoNT/A, BoNT/C1 or BoNT/E substrate of the invention can becleaved, for example, with an initial hydrolysis rate that is at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%,or 300% of the initial hydrolysis rate, under otherwise identicalconditions, of human SNAP-25 by BoNT/A, BoNT/C1 or BoNT/E, respectively,where the substrate of the invention and human SNAP-25 each is presentat a concentration of 1.0 mM. In other embodiments, a BoNT/A, BoNT/C1 orBoNT/E substrate of the invention is with an initial hydrolysis ratethat is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,150%, 200%, 250%, or 300% of the initial hydrolysis rate, underotherwise identical conditions, of human SNAP-25 by BoNT/A, BoNT/C1 orBoNT/E, respectively, where the substrate of the invention and humanSNAP-25 each is present at a concentration of 50 mM.

Similarly, a BoNT/B, BoNT/D, BoNT/F or BoNT/G substrate of the inventioncan be cleaved, for example, with an initial hydrolysis rate that is atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,250%, or 300% of the initial hydrolysis rate, under otherwise identicalconditions, of human VAMP-2 by BoNT/B, BoNT/D, BoNT/F or BoNT/G,respectively, where substrate of the invention and human VAMP-2 each ispresent at a concentration of 1.0 mM. In other embodiments, a BoNT/B,BoNT/D, BoNT/F or BoNT/G substrate of the invention is cleaved with aninitial hydrolysis rate that is at least 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of the initialhydrolysis rate, under otherwise identical conditions, of human VAMP-2by BoNT/B, BoNT/D, BoNT/F or BoNT/G, respectively, where substrate ofthe invention and human VAMP-2 each is present at a concentration of 50mM.

The invention also provides a BoNT/C1 substrate of the invention that iscleaved with an initial hydrolysis rate that is at least 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% ofthe initial hydrolysis rate, under otherwise identical conditions, ofhuman syntaxin by BoNT/C1, where the BoNT/C1 substrate and humansyntaxin each is present at a concentration of 1.0 mM. In otherembodiments, the invention provides a BoNT/C1 substrate that is cleavedwith an initial hydrolysis rate that is at least 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of the initialhydrolysis rate, under otherwise identical conditions, of human syntaxinby BoNT/C1, where the BoNT/C1 substrate and human syntaxin each ispresent at a concentration of 50 mM.

The “turnover number,” or k_(cat), is the rate of breakdown of atoxin-substrate complex. A clostridial toxin substrate of the inventioncan be cleaved with a k_(cat) that is reduced or enhanced as compared tothe k_(cat) of human SNAP-25, human VAMP-2 or human syntaxin targetproteins when cleaved by the same clostridial toxin under the sameconditions. A clostridial toxin substrate of the invention such as aBoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNTsubstrate, can be cleaved, for example, with a k_(cat) of about 0.001 toabout 4000 sec⁻¹. In one embodiment, a clostridial toxin substrate ofthe invention such as a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F,BoNT/G or TeNT substrate is cleaved with a k_(cat) of about 1 to about4000 sec⁻¹. In other embodiments, a BoNT/A, BoNT/B, BoNT/C1, BoNT/D,BoNT/E, BoNT/F, BoNT/G or TeNT substrate of the invention has a k_(cat)of less than 5 sec⁻¹, 10 sec⁻¹, 25 sec⁻¹, 50 sec⁻¹, 100 sec⁻¹, 250sec⁻¹, 500 sec⁻¹, or 1000 sec⁻¹. A clostridial toxin substrate of theinvention such as a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F,BoNT/G or TeNT substrate also can have, for example, a k_(cat) in therange of 1 to 1000 sec⁻¹; 1 to 500 sec⁻¹; 1 to 250 sec⁻¹; 1 to 100sec⁻¹; 1 to 50 sec⁻¹; 10 to 1000 sec⁻¹; 10 to 500 sec⁻¹; 10 to 250sec⁻¹; 10 to 100 sec⁻¹; 10 to 50 sec⁻¹; 25 to 1000 sec⁻¹; 25 to 500sec⁻¹; 25 to 250 sec⁻¹; 25 to 100 sec⁻¹; 25 to 50 sec⁻¹; 50 to 1000sec⁻¹; 50 to 500 sec⁻¹; 50 to 250 sec⁻¹; 50 to 100 sec⁻¹; 100 to 1000sec⁻¹; 100 to 500 sec⁻¹; or 100 to 250 sec⁻¹. One skilled in the artunderstands the turnover number, k_(cat), is assayed under standardkinetic conditions in which there is an excess of substrate.

In particular embodiments, a clostridial toxin substrate of theinvention is a peptide or peptidomimetic. As used herein, the term“peptidomimetic” is used broadly to mean a peptide-like molecule that iscleaved by the same clostridial toxin as the peptide substrate uponwhich it is structurally based. Such peptidomimetics include chemicallymodified peptides, peptide-like molecules containing non-naturallyoccurring amino acids, and peptoids, which are peptide-like moleculesresulting from oligomeric assembly of N-substituted glycines, and arecleaved by the same clostridial toxin as the peptide substrate uponwhich the peptidomimetic is derived (see, for example, Goodman and Ro,Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry andDrug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages803-861).

A variety of peptidomimetics are known in the art including, forexample, peptide-like molecules which contain a constrained amino acid,a non-peptide component that mimics peptide secondary structure, or anamide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, for example, anα-methylated amino acid; an α,α-dialkylglycine or α-aminocycloalkanecarboxylic acid; an N^(α)—C^(α) cylized amino acid; an N^(α)-methylatedamino acid; a β- or γ-amino cycloalkane carboxylic acid; anα,β-unsaturated amino acid; a β,β-dimethyl or β-methyl amino acid; aβ-substituted-2,3-methano amino acid; an N—C^(δ) or C^(α)—C^(δ) cyclizedamino acid; or a substituted proline or another amino acid mimetic. Inaddition, a peptidomimetic which mimics peptide secondary structure cancontain, for example, a nonpeptidic β-turn mimic; γ-turn mimic; mimic ofβ-sheet structure; or mimic of helical structure, each of which is wellknown in the art. A peptidomimetic also can be a peptide-like moleculewhich contains, for example, an amide bond isostere such as aretro-inverso modification; reduced amide bond; methylenethioether ormethylenesulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and otherpeptidomimetics are encompassed within the meaning of the term“peptidomimetic” as used herein.

The invention provides, for example, a botulinum toxin serotype A(BoNT/A) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/A recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/A substrate of the invention can include, for example,at least six consecutive residues of SNAP-25, where the six consecutiveresidues include Gln-Arg, or a peptidomimetic thereof. Such a BoNT/Asubstrate also can have, for example, at least six consecutive residuesof human SNAP-25, where the six consecutive residues includeGln₁₉₇-Arg₁₉₈, or a peptidomimetic thereof. In one embodiment, a BoNT/Asubstrate of the invention includes the amino acid sequenceGlu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO: 1), or a peptidomimeticthereof. In another embodiment, a BoNT/A substrate of the inventionincludes residues 187 to 203 of human SNAP-25 (SEQ ID NO: 2), or apeptidomimetic thereof. A variety of donor fluorophores and acceptorsare useful in a BoNT/A substrate of the invention, including but notlimited to, fluorescein-tetramethylrhodamine, DABCYL-EDANS, and ALEXAFLUOR®-488-QSY® 7. Additional donor fluorophores and acceptors useful ina BoNT/A substrate of the invention are described further herein below.

As used herein, the term “botulinum toxin serotype A recognitionsequence” is synonymous with “BoNT/A recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/A under conditions suitable for clostridial toxin proteaseactivity. A scissile bond cleaved by BoNT/A can be, for example,Gln-Ala.

A variety of BoNT/A recognition sequences are well known in the art. ABoNT/A recognition sequence can have, for example, residues 134 to 206or residues 137 to 206 of human SNAP-25 (Ekong et al., supra, 1997; U.S.Pat. No. 5,962,637). A BoNT/A recognition sequence also can include,without limitation, the sequenceThr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ ID NO: 27), ora peptidomimetic thereof, which corresponds to residues 190 to 202 ofhuman SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO:28), or a peptidomimetic thereof, which corresponds to residues 187 to201 of human SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ IDNO: 29), or a peptidomimetic thereof, which corresponds to residues 187to 202 of human SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu (SEQID NO: 30), or a peptidomimetic thereof, which corresponds to residues187 to 203 of human SNAP-25;Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQID NO: 31), or a peptidomimetic thereof, which corresponds to residues186 to 202 of human SNAP-25; orAsp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu(SEQ ID NO: 32), or a peptidomimetic thereof, which corresponds toresidues 186 to 203 of human SNAP-25. See, for example, Schmidt andBostian, J. Protein Chem. 14:703-708 (1995); Schmidt and Bostian, supra,1997; Schmidt et al., FEBS Letters 435:61-64 (1998); and Schmidt andBostian, U.S. Pat. No. 5,965,699). If desired, a similar BoNT/Arecognition sequence can be prepared from a corresponding (homologous)segment of another BoNT/A-sensitive SNAP-25 isoform or homolog such as,for example, murine, rat, goldfish or zebrafish SNAP-25 or can be any ofthe peptides disclosed herein or described in the art, for example, inU.S. Pat. No. 5,965,699.

A BoNT/A recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype A, or can besubstantially similar to a segment of a BoNT/A-sensitive protein. Asillustrated in Table 2, a variety of naturally occurring proteinssensitive to cleavage by BoNT/A are known in the art and include, forexample, human, mouse and rat SNAP-25; and goldfish SNAP-25A andSNAP-25B. Thus, a BoNT/A recognition sequence useful in a BoNT/Asubstrate of the invention can correspond, for example, to a segment ofhuman SNAP-25, mouse SNAP-25, rat SNAP-25, goldfish SNAP-25A or 25B, oranother naturally occurring protein sensitive to cleavage by BoNT/A.Furthermore, comparison of native SNAP-25 amino acid sequences cleavedby BoNT/A reveals that such sequences are not absolutely conserved (seeTable 2 and FIG. 5), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/A-sensitive SNAP-25 sequence can be tolerated in a BoNT/A substrateof the invention.

TABLE 2 Cleavage of SNAP-25 and related proteins^(a,b,c,d) Resistance toSpecies - Isoform Cleavage Sites SEQ ID NO: Cleavage by   BoNT/E            BoNT/A   BoNT/C          

human ¹⁷⁴           ²⁰⁶ none^(a) mouse - SNAP-25 qnrqid ri mekadsnktridean qra tkmlgsg rat ¹⁸⁰           ^(end) all^(b)human - SNAP-23  qnpqik ri tdkadtnrdridian ara kklids ¹⁷⁹          ^(end) BoNT/A & C mouse - SNAP-23  qnqqiq ki tekadtnknridiantra kklids ¹⁷⁴           ^(end) BoNT/A & C chicken - SNAP-25 qnrqid ri meklipikpglmkpt svq qrcsavvk ¹⁷¹           ^(end) nonegoldfish - SNAP-25A  qnrqid ri mdmadsnktridean qra tkmlgsg ¹⁷²          ^(end) none goldfish - SNAP-25B  qnrqid ri mekadsnktrideanqra tkmlgsg ¹⁸⁰           ^(end) BoNT/E^(c )& A^(d) Torpedo - SNAP-25 qnaqvd ri vvkgdmnkaridean kha tkml ¹⁸⁰           ^(end) (?)^(e)sea urchin - SNAP-25  qnsqvg ri tskaesnegrinsad kra knilrnk ²⁰³          ^(end) BONT/A & C C-elegans - SNAP-25 qnrqld ri hdkqsnevrvesank rak nlitk ¹⁸²           ^(end) BoNT/E & A^(e)Drosophila - SNAP-25  qnrqid ri nrkgesneariavan qra hqllk ¹⁸¹          ^(end) BoNT/A^(e) leech - SNAP-25  qnrqvd ri nnkmtsnqlrisdankra skllke ^(a)= In vitro cleavage of SNAP-25 requires 1000-fold higherBoNT/C concentration than BoNT/A or /E. ^(b)= Substitution of p182r, ork185dd (boxes) induces susceptibility toward BoNT/E. ^(c)= Resistance toBoNT/A possibly due to d189 or e189 substitution by v189, see box. ^(d)=Note that Torpedo is susceptible to BoNT/A. ^(e)= Note the presence ofseveral non-conservative mutations around putative cleavage sites.

TABLE 3 Kinetic parameters of BoNT/A synthetic peptide substratesRelative Peptide Sequence^(a) SEQ ID NO: Rate^(b) [1-15] SNKTRIDEANQRATK28 0.03 [1-16] SNKTRIDEANQRATKM 29 1.17 [1-17] SNKTRIDEANQRATKML 30 1.00M16A SNKTRIDEANQRATK A L 44 0.38 M16X SNKTRIDEANQRATK X L 45 1.20 K15ASNKTRIDEANQRAT A ML 46 0.12 T14S SNKTRIDEANQRA S KML 47 0.26 T14BSNKTRIDEANQRA B KML 48 1.20 A13B SNKTRIDEANQR B TKML 49 0.79 Q11ASNKTRIDEAN A RATKML 50 0.19 Q11B SNKTRIDEAN B RATKML 51 0.25 Q11NSNKTRIDEAN N RATKML 52 0.66 N10A SNKTRIDEA A QRATKML 53 0.06 A9BSNKTRIDE B NQRATKML 54 0.38 E8Q SNKTRID Q ANQRATKML 55 2.08 D7N SNKTRI NEANQRATKML 56 0.23 ^(a)Nonstandard amino acid abbreviations are: B,2-aminobutyric acid; X, 2-aminohexanoic acid (norleucine) ^(b)Initialhydrolysis rates relative to peptide [1-17]. Peptide concentrations were1.0 mM.

A clostridial toxin substrate of the invention, such as a BoNT/Asubstrate, can have one or multiple modifications as compared to anaturally occurring sequence that is cleaved by the correspondingclostridial toxin. For example, as compared to a 17-mer corresponding toresidues 187 to 203 of human SNAP-25, substitution of Asp193 with Asn inthe BoNT/A substrate resulted in a relative rate of proteolysis of 0.23;substitution of Glu194 with Gln resulted in a relative rate of 2.08;substitution of Ala195 with 2-aminobutyric acid resulted in a relativerate of 0.38; and substitution of Gln197 with Asn, 2-aminobutyric acidor Ala resulted in a relative rate of 0.66, 0.25, or 0.19, respectively(see Table 3). Furthermore, substitution of Ala199 with 2-aminobutyricacid resulted in a relative rate of 0.79; substitution of Thr200 withSer or 2-aminobutyric acid resulted in a relative rate of 0.26 or 1.20,respectively; substitution of Lys201 with Ala resulted in a relativerate of 0.12; and substitution of Met202 with Ala or norleucine resultedin a relative rate of 0.38 or 1.20, respectively. See Schmidt andBostian, supra, 1997. These results indicate that a variety of residuescan be substituted in a clostridial toxin substrate of the invention ascompared to a naturally occurring toxin-sensitive sequence. In the caseof BoNT/A, these results indicate that residues including but notlimited to Glu194, Ala195, Gln197, Ala199, Thr200 and Met202, Leu203,Gly204, Ser205, and Gly206, as well as residues more distal from theGln-Arg scissile bond can be substituted or can be conjugated to a donorfluorophore or acceptor to produce a BoNT/A substrate of the invention.Such a BoNT/A substrate is detectably proteolyzed at the scissile bondby BoNT/A under conditions suitable for clostridial toxin proteaseactivity. Thus, a BoNT/A substrate of the invention can include, ifdesired, one or several amino acid substitutions, additions or deletionsrelative to a naturally occurring SNAP-25 sequence.

In standard nomenclature, the sequence surrounding clostridial toxincleavage sites is denoted P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′, withP₁-P₁′ the scissile bond. In one embodiment, the invention provides aBoNT/A substrate or other clostridial toxin substrate in which theresidue at position P₁, P₂, P₃, P₄, P₅, or P_(>5) is substituted with anamino acid conjugated to a donor fluorophore or acceptor, and in whichthe residue at position P₁′, P₂′, P₃′, P₄′, P₅′ or P_(>5)′ issubstituted with an amino acid conjugated to a donor fluorophore oracceptor. In another embodiment, the invention provides a BoNT/Asubstrate or other clostridial toxin substrate in which the residue atposition P₁, P₃, P₄ or P_(>5) is substituted with an amino acidconjugated to a donor fluorophore or acceptor, and in which the residueat position P₂′, P₃′, P₅′ or P_(>5)′ is substituted with an amino acidconjugated to a donor fluorophore or acceptor. It is further understoodthat the amino acid side chain of the residue conjugated to a donorfluorophore or acceptor can be otherwise identical to the residuepresent in the corresponding position of the naturally occurring targetprotein, or can contain, for example, a different side chain. Furtherprovided by the invention is a BoNT/A substrate or other clostridialtoxin substrate in which the residue at P₃, P₄ or P_(>5) is substitutedwith an amino acid conjugated to a donor fluorophore or acceptor, and inwhich the residue at position P₂′, P₃′, P₅′ or P_(>5)′ is substitutedwith an amino acid conjugated to a donor fluorophore or acceptor. Again,the amino acid side chain of the residue conjugated to a donorfluorophore or acceptor can be otherwise identical to the residuepresent in the corresponding position of the naturally occurring targetprotein, or can contain, for example, a different side chain.

A BoNT/A substrate of the invention also can include, if desired, acarboxy-terminal amide. Thus, a BoNT/A substrate of the invention canbe, for example, a peptide having at most twenty, thirty, forty or fiftyresidues and containing a carboxy-terminal amide.

Further provided by the invention is a botulinum toxin serotype B(BoNT/B) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/B recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/B substrate of the invention can contain, for example,at least six consecutive residues of VAMP, where the six consecutiveresidues include Gln-Phe, or a peptidomimetic thereof. For example, aBoNT/B substrate of the invention can contain at least six consecutiveresidues of human VAMP-2, the six consecutive residues includingGln₇₆-Phe₇₇, or a peptidomimetic thereof. In one embodiment, a BoNT/Bsubstrate includes the amino acid sequenceGly-Ala-Ser-Gln-Phe-Glu-Thr-Ser (SEQ ID NO: 3), or a peptidomimeticthereof. In other embodiments, a BoNT/B substrate includes residues 55to 94 of human VAMP-2 (SEQ ID NO: 4); residues 60 to 94 of human VAMP-2(SEQ ID NO: 4); or residues 60 to 88 of human VAMP-2 (SEQ ID NO: 4), ora peptidomimetic of one of these sequences. It is understood that avariety of donor fluorophores and acceptors are useful in a BoNT/Bsubstrate of the invention; such donor fluorophore-acceptor combinationsinclude, but are not limited to, fluorescein-tetramethylrhodamine;DABCYL-EDANS; and ALEXA FLUOR®-488-QSY® 7. A variety of additional donorfluorophores and acceptors useful in a BoNT/B substrate of the inventionare known in the art and described further below.

As used herein, the term “botulinum toxin serotype B recognitionsequence” is synonymous with “BoNT/B recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/B under appropriate conditions. A scissile bond cleaved by BoNT/Bcan be, for example, Gln-Phe.

A variety of BoNT/B recognition sequences are well known in the art orcan be defined by routine methods. Such a BoNT/B recognition sequencecan include, for example, a sequence corresponding to some or all of thehydrophilic core of a VAMP protein such as human VAMP-1 or human VAMP-2.A BoNT/B recognition sequence can include, without limitation, residues33 to 94, residues 45 to 94, residues 55 to 94, residues 60 to 94,residues 65 to 94, residues 60 to 88 or residues 65 to 88 of humanVAMP-2 (SEQ ID NO: 4), or residues 60 to 94 of human VAMP-1 (SEQ ID NO:96) (see, for example, Shone et al., Eur. J. Biochem. 217: 965-971(1993) and U.S. Pat. No. 5,962,637). If desired, a similar BoNT/Brecognition sequence can be prepared from a corresponding (homologous)segment of another BoNT/B-sensitive VAMP isoform or homolog such ashuman VAMP-1 or rat or chicken VAMP-2.

Thus, it is understood that a BoNT/B recognition sequence can correspondto a segment of a protein that is sensitive to cleavage by botulinumtoxin serotype B, or can be substantially similar to such a segment of aBoNT/B-sensitive protein. As shown in Table 4, a variety of naturallyoccurring proteins sensitive to cleavage by BoNT/B are known in the artand include, for example, human, mouse and bovine VAMP-1 and VAMP-2; ratVAMP-2; rat cellubrevin; chicken VAMP-2; Torpedo VAMP-1; sea urchinVAMP; Aplysia VAMP; squid VAMP; C. elegans VAMP; Drosophila n-syb; andleech VAMP. Thus, a BoNT/B recognition sequence useful in a BoNT/Bsubstrate of the invention can correspond, for example, to a segment ofhuman VAMP-1 or VAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2,rat VAMP-2, rat cellubrevin, chicken VAMP-2, Torpedo VAMP-1, sea urchinVAMP, Aplysia VAMP, squid VAMP, C. elegans VAMP, Drosophila n-syb, leechVAMP, or another naturally occurring protein sensitive to cleavage byBoNT/B. Furthermore, as shown in Table 4, comparison of native VAMPamino acid sequences cleaved by BoNT/B reveals that such sequences arenot absolutely conserved (see, also, FIG. 6), indicating that a varietyof amino acid substitutions and modifications relative to a naturallyoccurring VAMP sequence can be tolerated in a BoNT/B substrate of theinvention.

TABLE 4 Cleavage of VAMP^(a,b) Resistance to Species - IsoformCleavage Sites SEQ ID NO: to Cleavage                         BoNT/B  BoNT/F  BoNT/D       TeNT         BoNT/G         

      

human ⁵³                   ⁹² none mouse - VAMP-1dkvlerd qkl selddradalqagas qf ess aa klkrkyww bovine human ⁵¹                  ⁹⁰ none mouse - VAMP-2 dkvlerd qkl selddradalqagasqf ets aa klkrkyww bovine ⁵³                   ⁹² TeNT & rat - VAMP-2dkvlerd qkl selddradalqagas vf ess aa klkrkyww BoNT/B ⁵¹                  ⁹⁰ none rat - VAMP-2 dkvlerd qkl selddradalqagasqf ets aa klkrkyww ³⁸                   ⁷⁷ none rat - Cellubrevindkvlerd qkl selddradalqagas qf ets aa klkrkyww ¹⁴⁶                   ¹⁷⁵all rat - TI-VAMP dlvaqrg erl ellidktenlvdssv tf ktt sr nlaramcm ⁻                  ⁻ TeNT & chicken - VAMP-1 ----erd qkl selddradalqagasvf ess aa klkr---- BoNT/B ⁻                   ⁻ none chicken - VAMP-2----erd qkl selddradalqagas qf ets aa klkr--- ⁵⁵                   ⁹⁴none Torpedo - VAMP-1 dkvlerd qkl selddradalqagas qf ess aa klkrkyww ³⁵                  ⁷⁴ BoNT/F, D & sea urchin - VAMPdkvldrd qal svlddradalqqgas qf etn ag klkrkyww G ⁴¹                   ⁸⁰BoNT/G Aplysia - VAMP ekvldrd qki sqlddraealqagas qf eas ag klkrkyww ⁶⁰                  ⁹⁹ BoNT/F & G squid - VAMP dkvlerd ski selddradalqagasqf eas ag klkrkfww ⁸⁶                   ¹¹⁵ BoNT F, D & C. elegans -VAMP nkvmerd vql nsldhraevlqngas qf qqs sr elkrqyww G ⁶⁷                  ¹⁰⁶ TeNT & Drosphila - syb^(a)ekvlerd qkl selgeradqleqgas qs eqq ag klkrkqww BoNT/B & G ⁶¹                  ¹⁰⁰ BoNT F & G Drosphila - n-sy^(b)ekvlerd skl selddradalqqgas qf eqq ag klkrkfwl ⁴⁹                   ⁸⁸BoNT/G leech - VAMP dkvlekd qkl aeldgradalqagas qf eas ag klkrkfww ^(a)=Sequence corrected in position 93 (f > s). ^(b)= Sequence corrected inposition 68 (t > s).

The invention also provides a botulinum toxin serotype C1 (BoNT/C1)substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/C1 recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/C1 substrate of the invention can have, for example, atleast six consecutive residues of syntaxin, the six consecutive residuesincluding Lys-Ala, or a peptidomimetic thereof. For example, a BoNT/C1substrate of the invention can have at least six consecutive residues ofhuman syntaxin, the six consecutive residues including Lys₂₅₃-Ala₂₅₄, ora peptidomimetic thereof. In one embodiment, a BoNT/C1 substratecontains the amino acid sequence Asp-Thr-Lys-Lys-Ala-Val-Lys-Tyr (SEQ IDNO: 5), or a peptidomimetic thereof.

A BoNT/C1 substrate of the invention also can contain, for example, atleast six consecutive residues of SNAP-25, where the six consecutiveresidues include Arg-Ala, or a peptidomimetic thereof. Such a BoNT/C1substrate can have, for example, at least six consecutive residues ofhuman SNAP-25, the six consecutive residues including Arg₁₉₈-Ala₁₉₉, ora peptidomimetic thereof. An exemplary BoNT/C1 substrate containsresidues 93 to 202 of human SNAP-25 (SEQ ID NO: 2), or a peptidomimeticthereof. As for all the clostridial toxin substrates of the invention, avariety of donor fluorophore-acceptor combinations are useful in aBoNT/C1 substrate, including but not limited to,fluorescein-tetramethylrhodamine; DABCYL-EDANS; and ALEXAFLUOR®-488-QSY® 7. Additional donor fluorophores and acceptors useful ina BoNT/C1 substrate of the invention are described herein below.

As used herein, the term “botulinum toxin serotype C1 recognitionsequence” is synonymous with “BoNT/C1 recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/C1 under appropriate conditions. A scissile bond cleaved by BoNT/C1can be, for example, Lys-Ala or Arg-Ala.

TABLE 5 Cleavage of syntaxin Resistance to Species IsoformCleavage Sites SEQ ID NO: Cleavage by        BoNT/C              

human ²⁴⁵                    ²⁶² no rat syntaxin 1A  eravsdtk ka vkyqskar mouse bovine human ²⁴⁴                    ²⁶¹ norat syntaxin 1B   eravsdtk ka vkyqskar mouse bovine²⁴⁵                    ²⁶² no rat syntaxin 2   ehakeetk ka ikyqskar²⁴⁴                    ²⁶¹ no rat syntaxin 3   ekardetr ka mkyqgqar²⁴⁴                    ²⁶¹ yes rat syntaxin 4   ergqehvk ia lenqkkar²³⁹                    ²⁵⁹ expected chicken syntaxin 1B  vpevfvtk sa vmyqcksr ²⁴³                    ²⁶⁰ no sea urchin syntaxin  vrrqndtk ka vkyqskar ²⁴⁷                    ²⁶⁴ no Aplysia syntaxin 1  etakmdtk ka vkyqskar ²⁴⁸                    ²⁶⁵ no squid syntaxin  etakvdtk ka vkyqskar ²⁴⁸                    ²⁶⁵ no Drosophila Dsynt 1  qtatqdtk ka lkyqskar ²⁵¹                    ²⁶⁸ no leech syntaxin 1  etaaadtk ka mkyqsaar

It is understood that a BoNT/C1 recognition sequence can correspond to asegment of a protein that is sensitive to cleavage by botulinum toxinserotype C1, or can be substantially similar to a segment of aBoNT/C1-sensitive protein. As shown in Table 5, a variety of naturallyoccurring proteins sensitive to cleavage by BoNT/C1 are known in the artand include, for example, human, rat, mouse and bovine syntaxin 1A and1B; rat syntaxins 2 and 3; sea urchin syntaxin; Aplysia syntaxin 1;squid syntaxin; Drosophila Dsynt1; and leech syntaxin 1. Thus, a BoNT/C1recognition sequence useful in a BoNT/C1 substrate of the invention cancorrespond, for example, to a segment of human, rat, mouse or bovinesyntaxin 1A or 1B, rat syntaxin 2, rat syntaxin 3, sea urchin syntaxin,Aplysia syntaxin 1, squid syntaxin, Drosophila Dsynt1, leech syntaxin 1,or another naturally occurring protein sensitive to cleavage by BoNT/C1.Furthermore, comparison of native syntaxin amino acid sequences cleavedby BoNT/C1 reveals that such sequences are not absolutely conserved (seeTable 5 and FIG. 7), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/C1-sensitive syntaxin sequence can be tolerated in a BoNT/C1substrate of the invention.

A variety of naturally occurring SNAP-25 proteins also are sensitive tocleavage by BoNT/C1, including human, mouse and rat SNAP-25; goldfishSNAP-25A and 25B; and Drosophila and leech SNAP-25. Thus, a BoNT/C1recognition sequence useful in a BoNT/C1 substrate of the invention cancorrespond, for example, to a segment of human, mouse or rat SNAP-25,goldfish SNAP-25A or 25B, Torpedo SNAP-25, zebrafish SNAP-25, DrosophilaSNAP-25, leech SNAP-25, or another naturally occurring protein sensitiveto cleavage by BoNT/C1. As discussed above in regard to variants ofnaturally occurring syntaxin sequences, comparison of native SNAP-25amino acid sequences cleaved by BoNT/C1 reveals significant sequencevariability (see Table 2 and FIG. 5 above), indicating that a variety ofamino acid substitutions and modifications relative to a naturallyoccurring BoNT/C1-sensitive SNAP-25 sequence can be tolerated in aBoNT/C1 substrate of the invention.

The present invention further provides a botulinum toxin serotype D(BoNT/D) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/D recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/D substrate of the invention can have, for example, atleast six consecutive residues of VAMP, the six consecutive residuesincluding Lys-Leu, or a peptidomimetic thereof. In one embodiment, aBoNT/D substrate contains at least six consecutive residues of humanVAMP, the six consecutive residues including Lys₅₉-Leu60, or apeptidomimetic thereof. In another embodiment, a BoNT/D substrate of theinvention contains the amino acid sequenceArg-Asp-Gln-Lys-Leu-Ser-Glu-Leu (SEQ ID NO: 6), or a peptidomimeticthereof. In a further embodiment, a BoNT/D substrate of the inventionincludes residues 27 to 116 of rat VAMP-2 (SEQ ID NO: 7), or apeptidomimetic thereof. It is understood that a variety of donorfluorophore-acceptor combinations are useful in a BoNT/D substrate ofthe invention; such donor fluorophore-acceptor pairs include, but arenot limited to, fluorescein-tetramethylrhodamine; DABCYL-EDANS; andALEXA FLUOR®-488-QSY® 7. Additional exemplary donor fluorophores andacceptors useful in a BoNT/D substrate of the invention are providedherein below.

The term “botulinum toxin serotype D recognition sequence” is synonymouswith “BoNT/D recognition sequence” and means a scissile bond togetherwith adjacent or non-adjacent recognition elements sufficient fordetectable proteolysis at the scissile bond by a BoNT/D underappropriate conditions. A scissile bond cleaved by BoNT/D can be, forexample, Lys-Leu.

A variety of BoNT/D recognition sequences are well known in the art orcan be defined by routine methods. A BoNT/D recognition sequence caninclude, for example, residues 27 to 116; residues 37 to 116; residues 1to 86; residues 1 to 76; or residues 1 to 69 of rat VAMP-2 (SEQ ID NO:7; Yamasaki et al., J. Biol. Chem. 269:12764-12772 (1994)). Thus, aBoNT/D recognition sequence can include, for example, residues 27 to 69or residues 37 to 69 of rat VAMP-2 (SEQ ID NO: 7). If desired, a similarBoNT/D recognition sequence can be prepared from a corresponding(homologous) segment of another BoNT/D-sensitive VAMP isoform or homologsuch as human VAMP-1 or human VAMP-2.

A BoNT/D recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype D, or can besubstantially similar to a segment of a BoNT/D-sensitive protein. Asshown in Table 5, a variety of naturally occurring proteins sensitive tocleavage by BoNT/D are known in the art and include, for example, human,mouse and bovine VAMP-1 and VAMP-2; rat VAMP-1 and VAMP-2; ratcellubrevin; chicken VAMP-1 and VAMP-2; Torpedo VAMP-1; Aplysia VAMP;squid VAMP; Drosophila syb and n-syb; and leech VAMP. Thus, a BoNT/Drecognition sequence useful in a BoNT/D substrate of the invention cancorrespond, for example, to a segment of human VAMP-1 or VAMP-2, mouseVAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, rat VAMP-1 or VAMP-2, ratcellubrevin, chicken VAMP-1 or VAMP-2, Torpedo VAMP-1, Aplysia VAMP,squid VAMP, Drosophila syb or n-syb, leech VAMP, or another naturallyoccurring protein sensitive to cleavage by BoNT/D. Furthermore, as shownin Table 5 above, comparison of native VAMP amino acid sequences cleavedby BoNT/D reveals significant sequence variability (see, also, FIG. 6),indicating that a variety of amino acid substitutions and modificationsrelative to a naturally occurring BoNT/D-sensitive VAMP sequence can betolerated in a BoNT/D substrate of the invention.

The present invention additionally provides a botulinum toxin serotype E(BoNT/E) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/E recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/E substrate can contain, for example, at least sixconsecutive residues of SNAP-25, the six consecutive residues includingArg-Ile, or a peptidomimetic thereof. Such a BoNT/E substrate can have,for example, at least six consecutive residues of human SNAP-25, the sixconsecutive residues including Arg₁₈₀-Ile₁₈₁, or a peptidomimeticthereof. In one embodiment, a BoNT/E substrate includes the amino acidsequence Gln-Ile-Asp-Arg-Ile-Met-Glu-Lys (SEQ ID NO: 8), or apeptidomimetic thereof. In another embodiment, a BoNT/E substrateincludes residues 156 to 186 of human SNAP-25 (SEQ ID NO: 2), or apeptidomimetic thereof. A variety of donor fluorophore-acceptorcombinations are useful in a BoNT/E substrate of the invention. Thesedonor fluorophore-acceptor combinations include, without limitation,fluorescein-tetramethylrhodamine, DABCYL-EDANS, ALEXA FLUOR®-488-QSY® 7,and additional donor fluorophores and acceptors described further below.

As used herein, the term “botulinum toxin serotype E recognitionsequence” is synonymous with “BoNT/E recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/E under appropriate conditions. A scissile bond cleaved by BoNT/Ecan be, for example, Arg-Ile.

One skilled in the art appreciates that a BoNT/E recognition sequencecan correspond to a segment of a protein that is sensitive to cleavageby botulinum toxin serotype E, or can be substantially similar to asegment of a BoNT/E-sensitive protein. A variety of naturally occurringproteins sensitive to cleavage by BoNT/E are known in the art andinclude, for example, human, mouse and rat SNAP-25; mouse SNAP-23;chicken SNAP-25; goldfish SNAP-25A and SNAP-25B; zebrafish SNAP-25; C.elegans SNAP-25; and leech SNAP-25 (see Table 2). Thus, a BoNT/Erecognition sequence useful in a BoNT/E substrate of the invention cancorrespond, for example, to a segment of human SNAP-25, mouse SNAP-25,rat SNAP-25, mouse SNAP-23, chicken SNAP-25, goldfish SNAP-25A or 25B,C. elegans SNAP-25, leech SNAP-25, or another naturally occurringprotein sensitive to cleavage by BoNT/E. Furthermore, as shown in Table2 and FIG. 5 above, comparison of native SNAP-23 and SNAP-25 amino acidsequences cleaved by BoNT/E reveals that such sequences are notabsolutely conserved, indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/E-sensitive SNAP-23 or SNAP-25 sequence can be tolerated in aBoNT/E substrate of the invention.

The invention also provides a botulinum serotype A/E (BoNT/A/E)substrate containing (a) a donor fluorophore; (b) an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and (c) a BoNTA or BoNT/E recognition sequence containing acleavage site, where the cleavage site intervenes between the donorfluorophore and the acceptor and where, under the appropriateconditions, resonance energy transfer is exhibited between the donorfluorophore and the acceptor. As used herein, the term “botulinumserotype A/E substrate” or “BoNT/A/E substrate” or “A/E substrate” meansa substrate that is susceptible to cleavage either by a botulinumserotype A toxin or a botulinum serotype E toxin. Such a botulinumserotype A/E substrate also can be susceptible to cleavage by both theBoNT/A and BoNT/E toxins. Any of the BoNT/A or BoNT/E recognitionsequences described herein or known in the art are useful in a BoNT/A/Esubstrate of the invention.

Further provided by the invention is a botulinum toxin serotype F(BoNT/F) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/F recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. Such a BoNT/F substrate can have, for example, at least sixconsecutive residues of VAMP, the six consecutive residues includingGln-Lys, or a peptidomimetic thereof. In one embodiment, a BoNT/Fsubstrate has at least six consecutive residues of human VAMP, the sixconsecutive residues including Gln₅₈-Lys₅₉, or a peptidomimetic thereof.In another embodiment, a BoNT/F substrate of the invention includesresidues 27 to 116 of rat VAMP-2 (SEQ ID NO: 7), or a peptidomimeticthereof. In a further embodiment, a BoNT/F substrate includes the aminoacid sequence Glu-Arg-Asp-Gln-Lys-Leu-Ser-Glu (SEQ ID NO: 9), or apeptidomimetic thereof. Those skilled in the art of fluorescenceresonance energy transfer understand that a variety of donorfluorophore-acceptor combinations are useful in a BoNT/F substrate ofthe invention. Non-limiting examples of donor fluorophore-acceptor pairsuseful in a BoNT/F substrate of the invention includefluorescein-tetramethylrhodamine, DABCYL-EDANS, ALEXA FLUOR®-488-QSY® 7,as well as additional donor fluorophore-acceptors combinations describedfurther below.

The term “botulinum toxin serotype F recognition sequence,” as usedherein, is synonymous with “BoNT/F recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/F under appropriate conditions. A scissile bond cleaved by BoNT/Fcan be, for example, Gln-Lys.

A variety of BoNT/F recognition sequences are well known in the art orcan be defined by routine methods. A BoNT/F recognition sequence caninclude, for example, residues 27 to 116; residues 37 to 116; residues 1to 86; residues 1 to 76; or residues 1 to 69 of rat VAMP-2 ((SEQ ID NO:7; Yamasaki et al., supra, 1994). A BoNT/F recognition sequence also caninclude, for example, residues 27 to 69 or residues 37 to 69 of ratVAMP-2 (SEQ ID NO: 7). It is understood that a similar BoNT/Frecognition sequence can be prepared, if desired, from a corresponding(homologous) segment of another BoNT/F-sensitive VAMP isoform or homologsuch as human VAMP-1 or human VAMP-2.

A BoNT/F recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype F, or can besubstantially similar to a segment of a BoNT/F-sensitive protein. Avariety of naturally occurring proteins sensitive to cleavage by BoNT/Fare known in the art and include, for example, human, mouse and bovineVAMP-1 and VAMP-2; rat VAMP-1 and VAMP-2; rat cellubrevin; chickenVAMP-1 and VAMP-2; Torpedo VAMP-1; Aplysia VAMP; Drosophila syb; andleech VAMP (see Table 5). Thus, a BoNT/F recognition sequence useful ina BoNT/F substrate of the invention can correspond, for example, to asegment of human VAMP-1 or VAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1or VAMP-2, rat VAMP-1 or VAMP-2, rat cellubrevin, chicken VAMP-1 orVAMP-2, Torpedo VAMP-1, Aplysia VAMP, Drosophila syb, leech VAMP, oranother naturally occurring protein sensitive to cleavage by BoNT/F.Furthermore, as shown in Table 5 above, comparison of native VAMP aminoacid sequences cleaved by BoNT/F reveals that such sequences are notabsolutely conserved (see, also, FIG. 6), indicating that a variety ofamino acid substitutions and modifications relative to a naturallyoccurring BoNT/F-sensitive VAMP sequence can be tolerated in a BoNT/Fsubstrate of the invention.

The present invention also provides a botulinum toxin serotype G(BoNT/G) substrate containing a donor fluorophore; an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore; and a BoNT/G recognition sequence that includes a cleavagesite, where the cleavage site intervenes between the donor fluorophoreand the acceptor and where, under the appropriate conditions, resonanceenergy transfer is exhibited between the donor fluorophore and theacceptor. A BoNT/G substrate can have, for example, at least sixconsecutive residues of VAMP, the six consecutive residues includingAla-Ala, or a peptidomimetic thereof. Such a BoNT/G substrate can have,for example, at least six consecutive residues of human VAMP, the sixconsecutive residues including Ala₈₃-Ala₈₄, or a peptidomimetic thereof.In one embodiment, a BoNT/G substrate contains the amino acid sequenceGlu-Thr-Ser-Ala-Ala-Lys-Leu-Lys (SEQ ID NO: 10), or a peptidomimeticthereof. As discussed above in regard to other clostridial toxinsubstrates, a variety of donor fluorophore-acceptor combinations areuseful in a BoNT/G substrate of the invention including for example,fluorescein-tetramethylrhodamine, DABCYL-EDANS, ALEXA FLUOR®-488-QSY® 7,and other donor fluorophore-acceptor combinations disclosed herein belowor well known in the art.

As used herein, the term “botulinum toxin serotype G recognitionsequence” is synonymous with “BoNT/G recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements sufficient for detectable proteolysis at the scissile bond by aBoNT/G under appropriate conditions. A scissile bond cleaved by BoNT/Gcan be, for example, Ala-Ala.

A BoNT/G recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype G, or can besubstantially similar to such a BoNT/G-sensitive segment. Asillustration in Table 5 above, a variety of naturally occurring proteinssensitive to cleavage by BoNT/G are known in the art and include, forexample, human, mouse and bovine VAMP-1 and VAMP-2; rat VAMP-1 andVAMP-2; rat cellubrevin; chicken VAMP-1 and VAMP-2; and Torpedo VAMP-1.Thus, a BoNT/G recognition sequence useful in a BoNT/G substrate of theinvention can correspond, for example, to a segment of human VAMP-1 orVAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, rat VAMP-1 orVAMP-2, rat cellubrevin, chicken VAMP-1 or VAMP-2, Torpedo VAMP-1, oranother naturally occurring protein sensitive to cleavage by BoNT/G.Furthermore, as shown in Table 5 above, comparison of native VAMP aminoacid sequences cleaved by BoNT/G reveals that such sequences are notabsolutely conserved (see, also, FIG. 6), indicating that a variety ofamino acid substitutions and modifications relative to a naturallyoccurring BoNT/G-sensitive VAMP sequence can be tolerated in a BoNT/Gsubstrate of the invention.

Also provided by the invention is a tetanus toxin (TeNT) substratecontaining a donor fluorophore; an acceptor having an absorbancespectrum overlapping the emission spectrum of the donor fluorophore; anda TeNT recognition sequence that includes a cleavage site, where thecleavage site intervenes between the donor fluorophore and the acceptorand where, under the appropriate conditions, resonance energy transferis exhibited between the donor fluorophore and the acceptor.

A TeNT substrate of the invention can have, for example, at least sixconsecutive residues of VAMP, the six consecutive residues includeGln-Phe, or a peptidomimetic thereof. For example, such a TeNT substratecan have at least six consecutive residues of human VAMP-2, the sixconsecutive residues including Gln₇₆-Phe₇₇, or a peptidomimetic thereof.In one embodiment, a TeNT substrate contains the amino acid sequenceGly-Ala-Ser-Gln-Phe-Glu-Thr-Ser (SEQ ID NO: 11), or a peptidomimeticthereof. In another embodiment, the TeNT substrate contains residues 33to 94 of human VAMP-2 (SEQ ID NO: 4); residues 25 to 93 of human VAMP-2(SEQ ID NO: 4); or residues 27 to 116 of rat VAMP-2 (SEQ ID NO: 7), or apeptidomimetic of one of these sequences. A variety of donorfluorophore-acceptor combinations are useful in a TeNT substrate of theinvention, including, without limitation,fluorescein-tetramethylrhodamine; DABCYL-EDANS; and ALEXAFLUOR®-488-QSY® 7. It is recognized that additional donor fluorophoresand acceptors, including those described further below, can be useful ina TeNT substrate of the invention.

The term “tetanus toxin recognition sequence” means a scissile bondtogether with adjacent or non-adjacent recognition elements sufficientfor detectable proteolysis at the scissile bond by a tetanus toxin underappropriate conditions. A scissile bond cleaved by TeNT can be, forexample, Gln-Phe.

A variety of TeNT recognition sequences are well known in the art or canbe defined by routine methods and include a sequence corresponding tosome or all of the hydrophilic core of a VAMP protein such as humanVAMP-1 or human VAMP-2. ATeNT recognition sequence can include, forexample, residues 25 to 93 or residues 33 to 94 of human VAMP-2 (SEQ IDNO: 4; Cornille et al., Eur. J. Biochem. 222:173-181 (1994); Foran etal., Biochem. 33: 15365-15374 (1994)); residues 51 to 93 or residues 1to 86 of rat VAMP-2 (SEQ ID NO: 7; Yamasaki et al., supra, 1994); orresidues 33 to 94 of human VAMP-1 (SEQ ID NO: 96). A TeNT recognitionsequence also can include, for example, residues 25 to 86, residues 33to 86 or residues 51 to 86 of human VAMP-2 (SEQ ID NO: 4) or rat VAMP-2(SEQ ID NO: 7). It is understood that a similar TeNT recognitionsequence can be prepared, if desired, from a corresponding (homologous)segment of another TeNT-sensitive VAMP isoform or species homolog suchas human VAMP-1 or sea urchin or Aplysia VAMP.

Thus, a TeNT recognition sequence can correspond to a segment of aprotein that is sensitive to cleavage by tetanus toxin, or can besubstantially similar to a segment of a TeNT-sensitive protein. As shownin Table 5 above, a variety of naturally occurring proteins sensitive tocleavage by TeNT are known in the art and include, for example, human,mouse and bovine VAMP-1 and VAMP-2; rat VAMP-2; rat cellubrevin; chickenVAMP-2; Torpedo VAMP-1; sea urchin VAMP; Aplysia VAMP; squid VAMP; C.elegans VAMP; Drosophila n-syb; and leech VAMP. Thus, a TeNT recognitionsequence useful in a TeNT substrate of the invention can correspond, forexample, to a segment of human VAMP-1 or VAMP-2, mouse VAMP-1 or VAMP-2,bovine VAMP-1 or VAMP-2, rat VAMP-2, rat cellubrevin, chicken VAMP-2,Torpedo VAMP-1, sea urchin VAMP, Aplysia VAMP, squid VAMP, C. elegansVAMP, Drosophila n-syb, leech VAMP, or another naturally occurringprotein sensitive to cleavage by TeNT. Furthermore, comparison of nativeVAMP amino acid sequences cleaved by TeNT reveals that such sequencesare not absolutely conserved (Table 5 and FIG. 6), indicating that avariety of amino acid substitutions and modifications relative to anaturally occurring TeNT-sensitive VAMP sequence can be tolerated in aTeNT substrate of the invention.

The present invention relies, in part, on fluorescence resonance energytransfer (FRET), a physical process by which energy is transferrednon-radiatively from an excited donor fluorophore to an acceptor, whichmay be another fluorophore, through intramolecular long-rangedipole-dipole coupling. FRET is dependent on the inverse sixth power ofthe intramolecular separation of the donor fluorophore and acceptor, andfor effective transfer, the donor fluorophore and acceptor are in closeproximity, separated, for example, by about 10 A to about 100 A.Effective energy transfer is dependent on the spectral characteristicsof the donor fluorophore and acceptor as well as their relativeorientation. For effective transfer over 10 to 100 A, the quantum yieldof the donor fluorophore generally is at least 0.1, and the absorptioncoefficient of the acceptor generally is at least 1000 (see Clegg,Current Opinion in Biotech. 6:103-110 (1995); and Selvin, NatureStructural Biol. 7:730-734 (2000)).

In a clostridial toxin substrate of the invention, the donor fluorophoreand acceptor are selected so that the donor fluorophore and acceptorexhibit resonance energy transfer when the donor fluorophore is excited.One factor to be considered in choosing the donor fluorophore/acceptorpair is the efficiency of FRET between the donor fluorophore andacceptor. In one embodiment, the invention provides a clostridial toxinsubstrate in which, under optimal conditions, the efficiency of FRETbetween the donor fluorophore and acceptor is at least 10%. In anotherembodiment, the invention provides a clostridial toxin substrate inwhich, under optimal conditions, the efficiency of FRET between thedonor fluorophore and acceptor is at least 20%. In still furtherembodiments, the invention provides a clostridial toxin substrate inwhich, under optimal conditions, the efficiency of FRET between thedonor fluorophore and acceptor is at least 30%, 40%, 50%, 60%, 70% or80%.

As is well known in the art, the efficiency of FRET is dependent on theseparation distance and the orientation of the donor fluorophore andacceptor as described by the Förster equation, as well as thefluorescent quantum yield of the donor fluorophore and the energeticoverlap with the acceptor. In particular, the efficiency (E) of FRET canbe determined as follows:E=1−F _(DA) /F _(D)=1/(1+(R/R ₀)⁶)

-   -   where F_(DA) and F_(D) are the fluorescence intensities of the        donor fluorophore in the presence and absence of the acceptor,        respectively, and R is the distance between the donor        fluorophore and the acceptor.

The Förster radius (R_(o)) is the distance at which resonance energytransfer is 50% efficient, that is, 50% of excited donor fluorophoresare deactivated by FRET. The magnitude of the Förster radius depends onthe quantum yield of the donor fluorophore; the extinction coefficientof the acceptor; and the overlap between the donor fluorophore'semission spectrum and the acceptor's excitation spectrum.R _(O)=[8.8×10²³×κ² ×n ⁻⁴ ×QY _(D) ×J(λ)]^(1/6)Δ

-   -   where κ²=dipole orientation factor (range 0 to 4; κ²=⅔ for        randomly oriented donors and acceptors)    -   QY_(D)=fluorescence quantum yield of the donor in the absence of        the acceptor    -   n=refractive index    -   J(λ)=spectral overlap integral        -   =Iε_(.A)(λ)×F_(D)(λ)×λ⁴dλcm³M⁻¹    -   where ε_(.A)=extinction coefficient of acceptor    -   F_(D)=fluorescence emission intensity of donor as a fraction of        the total integrated intensity (Förster, Ann. Physik 2:55-75        (1948)).

Typical Förster radius values for various donor fluorophore/acceptorpairs are given in Table 6 below (see, also, Wu and Brand, AnalyticalBiochem. 218:1-13 (1994), which is incorporated herein by reference).Comprehensive lists of Förster radii also are known in the art (see, forexample, Berlman, Energy Transfer Parameters of Aromatic CompoundsAcademic Press, New York 1973). Furthermore, those skilled in the artrecognize that component factors of the Förster radius (R_(o)) aredependent upon the environment such that the actual value observed canvary from the listed value.

Any of a number of donor fluorophores and acceptors in variouscombinations can be useful in a clostridial toxin substrate of thepresent invention. A donor fluorophore generally is selected such thatthere is substantial spectral overlap between the emission spectrum ofthe donor fluorophore overlaps with the excitation spectrum of theacceptor. In addition, a donor fluorophore can be selected, for example,to have an excitation maximum near a laser frequency such asHelium-Cadmium 442 nm or argon 488 nm, whereby laser light serves as aneffective means to excite the donor fluorophore. In one embodiment, thewavelength maximum of the emission spectrum of the acceptor moiety is atleast 10 nm greater than the wavelength maximum of the excitationspectrum of the donor fluorophore. In a further embodiment, the acceptoris a fluorophore having an emission spectrum in the red portion of thevisible spectrum. In an additional embodiment, the acceptor is afluorophore having an emission spectrum in the infrared region of thespectrum. A variety of donor fluorophore-acceptor pairs, and theirFörster radii, are provided herein in Tables 6 and 7. See, also,Haugland, Handbook of Fluorescent Probes and Research Chemicals 6^(th)Edition, Molecular Probes, Inc., Eugene, Oreg., 1996, which isincorporated herein by reference.

TABLE 6 Donor fluorophore Acceptor Ro (Δ) Reference Fluorescein TMR49-54 Johnson et al., Biochemistry 32: 6402-6410 (1993); Odom et al.,Biochemistry 23: 5069- 5076 (1984) Fluorescein QSY ® 7 61 — EDANS DABCYL33 — Napthalene Dansyl 22 Haas et al., Proc. Natl. Acad. Sci. USA 72:1807-1811 (1975) IANBD DDPM 25 Kasprzyk et al., Biochemistry 22:1877-1882 (1983) IAEDANS DDPM 25-29 Dalbey et al., Biochemistry 22:4696-4706 (1983); Cheung et al., Biophys. Chem. 40: 1-17 (1991) DNSM LY26-32 Nalin et al., Biochemistry 28: 2318-2324 (1985) IAEDANS IANBD27-51 Franzen et al., Biochemistry 19: 6080-6089 (1980); First et al.,Biochemistry 28: 3606- 3613 (1989) ε-A _(F2)DNB 29 Perkins et al., J.Biol. Chem. 259: 8786-8793 (1984) Pyrene Bimane 30 Borochov-Neori andMontal, Biochemistry 28: 1711-1718 (1989) ANAI IPM 30 Peerce and Wright,Proc. Natl. Acad. Sci. USA 83: 8092-8096 (1986) IAANS IAF 31 Grossman,Biochim. Biophys. Acta 1040: 276-280 (1990) ε-A F₂DPS 31 Perkins et al.,supra, 1984 ε-A DDPM 31 Miki and Mihashi, Biochim. Biophys. Acta 533:163-172 (1978) IAEDANS TNP 31-40 Takashi et al., Biochemistry 21:5661-5668 (1982); dos Remedios and Cooke, Biochim. Biophys. Acta 788:193-205 (1984) MNA DACM 32 Amir and Haas, Biochemistry 26: 2162-2175(1987) PM NBD 32 Snyder and Hammes, Biochemistry 24: 2324-2331 (1985)FITC TNP-ATP 32 Amler et al., Biophys. J. 61: 553-568 (1992) DANZ DABM34 Albaugh and Steiner, J. Phys. Chem. 93: 8013-8016 (1989) NCP CPM 34Mitra and Hammes, Biochemistry 28: 3063-3069 (1989) NAA DNP 33-37McWherter et al., Biochemistry 25: 1951-1963 (1986) LY TNP-ATP 35 Nalin,supra, 1985 IAF dil-C₁₈ 35 Shahrokh et al., J. Biol. Chem. 266:12082-12089 (1991) IAF TMR 37 Taylor et al., J. Cell Biol. 89: 362-367(1981) FMA FMA 37 Dissing et al., Biochim. Biophys. Acta 553: 66-83(1979) PM DMAMS 38 Lin and Dowben, J. Biol. Chem. 258: 5142-5150 (1983)mBBR FITC 38 Tompa and Batke, Biochem. Int. 20: 487-494 (1990) mBBR DABM38 Kasprzak et al., Biochemistry 27: 4512-4523 (1988) ε-A NBD 38 Mikiand Iio, Biochim. Biophys. Acta 790: 201-207 (1984) Pyrene Coumarin 39Borochov-Neori and Montal, supra, 1989 IPM FNAI 39 Peerce and Wright,supra, 1986 IAEDANS DABM 40 Tao et al. Biochemistry 22: 3059-3066 (1983)IAEDANS TNP-ATP 40 Tao et al., supra, 1983 ε-A IANBD 40 Miki and Wahl,Biochim. Biophys. Acta 786: 188-196 (1984) NBD SRH 40-74 Wolf et al.,Biochemistry 31: 2865-2873 (1992) ISA TNP 42 Jacobson and Colman,Biochemistry 23: 3789-3799 (1984) Dansyl ODR 43 Lu et al., J. Biol.Chem. 264: 12956-12962 (1989) DANZ IAF 44-49 Cheung et al., Biochemistry21: 5135-5142 (1983) FNAI EITC 45 Peerce and Wright, supra, 1986 NBD LRH45-70 Wolf et al., supra, 1992 IAF EIA 46 Taylor et al., supra, 1981FITC ENAI 46 Peerce and Wright, supra, 1986 Proflavin ETSC 46 Robbins etal., Biochemistry 20: 5301-5309 (1981) CPM TNP-ATP 46 Snyder and Hammes,supra, 1985 IAEDANS IAF 46-56 Franzen, supra, 1985; Grossman, supra,1990 CPM Fluorescein 47 Thielen et al., Biochemistry 23: 6668-6674(1984) IAEDANS FITC 49 Jona et al., Biochim. Biophys. Acta 1028: 183-199(1990); Birmachu et al., Biochemistry 28: 3940-3947 (1989) IAF TMR 50Shahrokh et al., J. Biol. Chem. 266: 12082-12089 (1991) CF TR 51 Johnsonet al., supra, 1993 CPM TRS 51 Odom et al., supra, 1984 ε-A TNP-ATP 51dos Remedios and Cooke, supra, 1984 CPM FM 52 Odom et al., supra, 1984LY EM 53 Shapiro et al., J. Biol. Chem. 266: 17276-17285 (1991) FITCEITC 54 Carraway et al., J. Biol. Chem. 264: 8699-8707 (1989) IAEDANSDiO-C₁₄ 57 Shahrokh et al., supra, 1991 IAF ErITC 58 Amler et al.,supra, 1992 FITC EM 60 Kosk-Kosicka et al., J. Biol. Chem. 264:19495-19499 (1989) FITC ETSC 61-64 Robbins et al., supra, 1981 FITCErITC 62 Amler et al., supra, 1992 BPE CY5 72 Ozinskas et al., Anal.Biochem. 213: 264-270 (1993) Fluorescein Fluorescein 44 — BODIPY ® FLBODIPY ® FL 57 — ANAI, 2-anthracence N-acetylimidazole; BPE,B-phycoerythrin; CF, carboxyfluorescein succinimidyl ester; CPM,7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin; CY5,carboxymethylindocyanine-N-hydroxysuccinimidyl ester; dil-C₁₈,1,1′-dioctadecyl-3-3,3,3′,3′-tetramethyl-indocarbocyanine; diO-C₁₄,3,3′-ditetradecyloxacarbocyanine; DABM,4-dimethylaminophenylazo-phenyl-4′-maleimide; DACM,(7-(dimethylamino)coumarin-4-yl)-acetyl; DANZ, dansylaziridine; DDPM,N-(4-dimethylamino-3,5-dinitrophenyl)maleimide; DMAMS,dimethylamino-4-maleimidostilbene; DSMN,N-(2,5′-dimethoxystiben-4-yl)-maleimide; DNP, 2,4-dinitrophneyl; ε-A,1,N⁶-ethenoadenosine; EIA, 5-(iodoacetetamido)eosin; EITC,eosin-5-isothiocyanate; ENAI, eosin N-acETYLIMIDAZOLE; EM, eosinmaleimide; ErITC, erythrosin-5′-isothiocyanate; ETSC, eosinthiosemicarazide; F₂DNB, 1,5-difluro-2,4′-dinitrobenzene; F₂DPS,4,4′-difluoro-3,3′-dinitrophenylsulfone; FITC, fluoresceinthiosemicarbazide; IAANS,2-(4′-iodoacetamido)anilino)napthalene-6-sulfonic acid; IAEDANS,5-(2-((iodoacetyl)amino)ethyl)amino)-napthlene-1-sulfonic acid; IAF,5-iodoacetamidofluorescein; IANBD,N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole;IPM, 3(4-isothiocyanatophenyl)7-diethyl-4-amino-4-methylcoumarin; ISA,4-(iodoacetamido)salicylic acid; LRH, lissaminerhodamine; LY, Luciferyellow; mBBR, monobromobiamane; MNA, (2-methoxy-1-naphthyl)-methyl; NAA,2-napthoxyacetic acid; NBD, 7-nirto-2,1,3-benzoxadiazol-4-yl; NCP,N-cyclohexyl-N′-(1-pyrenyl)carbodiimide; ODR, octadecylrhodamine; PM,N-(1-pyrene)-maleimide; SRH, sulforhodamine; TMR, tetramethylrhodamine;TNP, trinitrophenyl; and TR, Texas Red

An aromatic amino acid such as tryptophan or tyrosine also can be adonor fluorophore useful in a clostridial toxin substrate of theinvention. Exemplary donor fluorophore-acceptor pairs in whichtryptophan or tyrosine is the donor fluorophore and relevant Försterdistances are shown in Table 7 below. Modified amino acids also can beuseful as donor fluorophores or acceptors in a clostridial toxinsubstrate of the invention. Such fluorescent or quenching modified aminoacids are known in the art and include, for example, the fluorescentamino acid L-pyrenylalanine (Pya) and the non-fluorescent acceptorp-nitrophenylalanine (Nop), as described, for example, in Anne et al.,Analytical Biochem. 291:253-261 (2001).

TABLE 7 Förster Distances Using Trp as a Donor Donor Acceptor R_(o) (Δ)Reference Trp Ru(III)(NH3)5 12-16 Recchia et al., Biochim. Biophys. Acta702: 105-111 (1982) Trp Nitrobenzoyl 16 Wiczk et al., J. Fluo 1: 273-286(1991) Trp Dansyl 21 Steinberg, Annu. Rev. Biochem. 40: 83-114 (1971)Trp IAEDANS 22 Matsumoto and Hammes, Biochemistry 14: 214-224 (1975) TrpANS 23 Conrad and Brand, Biochemistry 7: 777- 787 (1968) Trp Anthroyloxy24 Wiczk et al., supra, 1991 Trp TNB 24 Wu and Brand, Biochemistry 31:7939- 7947 (1992) Trp Anthroyl 25 Burgun et al., Arch. Biochem. Biophys.286: 394-401 (1991) Trp Tyr-NO₂ 26 Steiner et al., J. Fluo. 1: 15-22(1991) Trp Pyrene 28 Vekshin, Mol. Biol. 17: 827-832 (1983) Trp Heme 29Ladokhin et al., Proc. SPIE 1640: 562-569 (1992) Trp NBS 30 Wiczk etal., supra, 1991 Trp DNBS 33 Wiczk et al., supra, 1991 Trp DPH 40 LeDoan et al., Biochim. Biophys. Acta 735: 259-270 (1983)

In view of the above, it is understood that a variety of donorfluorophore/acceptor pairs can be useful in a clostridial toxinsubstrate of the invention. A donor fluorophore-acceptor pair useful inthe invention can be, for example, the donor fluorophore fluorescein incombination with ROX (6-carboxy-X-rhodamine; Applied Biosystems Divisionof Perkin-Elmer Corporation; Foster City, Calif.); TAMRA(N,N,N′,N′-tetramethyl-6-carboxy-rhodamine; Applied Biosystems);rhodamine; texas red or eosin. A donor fluorophore-acceptor pair usefulin the invention also can be, for example, the donor fluorophore cascadeblue with fluorescein as an acceptor; the donor fluorophore BODIPY®530/550 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-5-indacene incombination with BODIPY® 542/563(4,4-difluoro-5-p-methoxyphenyl-4-bora-3a,4a-diaza-S-indacene) as anacceptor; or BODIPY® 542/563(4,4-difluoro-5-p-methoxyphenyl-4-bora-3a,4a-diaza-S-indacene incombination with BODIPY® 564/570(4,4-difluoro-5-styryl-4-bora-3a,4a-diaza- S-indacene as an acceptor.The numbers following the name BODIPY® reflect the excitation andemission maxima of the molecule; BODIPY® compounds are commerciallyavailable from Molecular Probes (Eugene Oreg.).

In one embodiment, the donor fluorophore is fluorescein. In a furtherembodiment, a clostridial toxin substrate of the invention contains afluorescein as the donor fluorophore and tetramethylrhodamine as theacceptor. Such a substrate can be excited in the range of 480 to 505 nm,for example, at 488 nm or 492 nm, and emission detected at 520 nm(λ_(em) fluorescein), 585 nm (λ_(em) tetramethylrhodamine), or both.Prior to cleavage of the substrate at the clostridial toxin cleavagesite, the tetramethylrhodamine emission intensity is greater than thatof fluorescein; substrate cleavage results in a change in the ratio offluorescein to tetramethylrhodamine intensity. Cleavage generallyresults in fluorescein becoming the dominant emitting fluorophore.Methods for preparing proteins and peptides containing fluorescein andtetramethylrhodamine are well known in the art (see, for example,Matsumoto et al., Bioorganic & Medicinal Chemistry Letters 10:1857-1861(2000)).

A donor fluorophore useful in a substrate of the invention also can be,for example, EDANS (λ_(Ab) 340 nM, λ_(em) 490 nm), which can be combinedwith an acceptor such as DABCYL. Where DABCYL and EDANS are combined ina clostridial toxin substrate of the invention, energy is transferredfrom the EDANS donor fluorophore to the DABCYL acceptor in the intactsubstrate, resulting in quenching of EDANS emission fluorescence. Uponcleavage at the toxin cleavage site, fluorescence of the cleaved EDANSproduct is increased and can be restored, for example, to the free donorfluorophore level. Efficient fluorescence quenching in the intactsubstrate occurs as a result of favorable energetic overlap of the EDANSemission spectrum and the DABCYL absorbance spectrum, and the relativelylong excited state lifetime of the EDANS donor fluorophore (Wang et al.,Tetrahedron Lett. 31:6493-6496 (1991); Holskin et al., Anal. Biochem.226:148-155 (1995); and Wang et al., Anal. Biochem. 210:351-359 (1993)).

Dansyl (DNS or 5-dimethylaminonaphthalene-1-sulfonyl) also can be auseful as a donor fluorophore or acceptor in a substrate of theinvention. In one embodiment, a clostridial toxin substrate of theinvention contains dansyl as the donor fluorophore; a dansyl donor canbe combined, for example, with a nitrophenyl residue acceptor such asPhe(pNO2), which acts as a quencher when in proximity to the dansyldonor fluorophore. Substrates containing a dansyl donor fluorophore, forexample, in combination with a nitrophenyl residue can be prepared asdescribed, for example, in Florentin et al., Anal. Biochem. 141:62-69(1984) or Goudreau et al., Anal. Biochem. 219:87-95 (1994). In anotherembodiment, a clostridial toxin substrate contains dansyl as theacceptor. Adansyl acceptor can act as a quencher when combined, forexample, with a donor fluorophore such as Trp (λ_(ex) 290 nm, λ_(em) 360nm). In a substrate containing Trp and dansyl, Trp fluorescence can bequenched 60% by energy transfer to the dansyl group, and this quenchingcan be significantly reduced or abolished in the presence of toxinprotease activity at the toxin cleavage site (see, for example,Geoghegan et al., FEBS Letters 262:119-122 (1990)).

It is understood that donor-acceptor pairs having well-separatedemission maxima can be useful in the substrates and methods of theinvention; well-separated emission maxima allow altered acceptoremission to be detected without donor emission contamination. A donorfluorophore, or acceptor, or both, can emit, for example, in thefar-red, for example, greater than 650 nm. Such far-red emitting donorfluorophores and acceptors include cyanine dyes such as Cy5, Cy5.5 andCy7 (Selvin, supra, 2000). In one embodiment, the invention provides aclostridial toxin substrate containing Cy3 and Cy5 as the donorfluorophore-acceptor pair; Cy3 emits maximally as 570 nm and Cy5 emitsmaximally at 670 nm. Such cyanine dyes can be prepared bystraightforward synthesis, as described, for example, in Gruber et al.,Bioconj. Chem. 11:161-166 (2000).

A donor fluorophore useful in a clostridial toxin substrate of theinvention also can be, for example, a lanthanide atom, also known as arare-earth element. Lanthanides such as terbium (Tb), europium (Eu),dysprosium (Dy) and samarium (Sm) have sharply spiked wavelengths,millisecond lifetimes following an excitation pulse, are unpolarized,and have high quantum yields. A lanthanide donor fluorophore such as aterbium or europium chelate can be combined with a variety of acceptorsincluding organic dye acceptor. A Eu-chelate donor fluorophore can becombined, for example, with allophycocyanin (APC), and a Tb-chelatedonor fluorophore can be combined, for example, withtetramethylrhodamine. Background fluorescence due to direct excitationis eliminated temporally; the lifetimes of organic acceptors generallyare in the nanosecond range, while the sensitized emission follows thelifetime of the donor fluorophore and is on the order of microseconds tomilliseconds (see Selvin, supra, 2000). Thus, determination of resonanceenergy transfer can be initiated relatively late following excitation,after non-specific interfering fluorescence has faded away. Lanthanidechelates are well known in the art and are commercially available, forexample, from EG&G-Wallac (Turku, Finland).

A donor fluorophore useful in the invention also can be the well knownfluorophore (7-methoxycoumarin-4-yl)acetyl (Mca), which can be combinedwith an acceptor such as the quencher 2,4-dinitrophenyl (Dnp). See, forexample, Kakiuchi et al., J. Virol. Methods 80:77-84 (1999). When Mca iscombined with the appropriate quencher such as Dnp in a clostridialtoxin substrate of the invention, increased donor emission fluorescencefrom Mca (λ_(em) 393 nm) is detected upon cleavage at the clostridialtoxin cleavage site and is indicative of toxin protease activity.

A donor fluorophore useful in a clostridial toxin substrate of theinvention also can be, for example, a 2-aminobenzoyl (Abz) group, whichcan be combined, if desired, with a quencher such as 2,4-dinitrophenyl(Dnp). In an intact clostridial toxin substrate, the Dnp group quenches,by resonance energy transfer, the fluorescence of the Abz group;proteolytic cleavage of the substrate relieves quenching and results inan increase in fluorescence proportional to the concentration of thereleased Abz fragment. A clostridial toxin substrate containing, forexample, Abz at the amino-terminus and a Dnp-derivatized residue such aslysine can be prepared by routine methods as described, for example, inLe Bonniec et al., Biochemistry 35:7114-7122 (1996)).

A donor fluorophore or acceptor useful in a clostridial toxin substrateof the invention also can be an ALEXA FLUOR®-dye, commercially availablefrom Molecular Probes (Eugene, Oreg.). ALEXA FLUOR® dyes useful in theinvention include, for example, ALEXA FLUOR® 350, ALEXA FLUOR®-430,ALEXA FLUOR®-488, ALEXA FLUOR®-532, ALEXA FLUOR®-546, ALEXA FLUOR®-568,ALEXA FLUOR®-594, ALEXA FLUOR®-633, ALEXA FLUOR®-647, ALEXA FLUOR® 660and ALEXA FLUOR®-680.

A donor fluorophore or acceptor useful in the invention also can be agenetically encoded dye such as green fluorescence protein (GFP), bluefluorescence protein (BFP), cyan fluorescence protein (CFP), yellowfluorescence protein (YFP) or red fluorescence protein such as dsRed (BDBiosciences Clontech; Palo Alto, Calif.). Such genetically encoded donorfluorophores and acceptors are well known in the art as described, forexample, in Selvin, supra, 2000, and Mahajan et al., Chemistry andBiology 6:401-409 (1999). For example, CFP has an excitation maxima at433 nm and an emission maxima at 476 nm, and can be used as a donorfluorophore in combination with YFP as an acceptor (emission maxima at527 nm). If desired, BFP can be used as a donor fluorophore incombination with GFP as the acceptor, or CFP can be used as the donorfluorophore in combination with YFP as the acceptor. Additionalgenetically encoded donor fluorophores and acceptors including Aequorearelated fluorescent proteins are well known in the art, as described,for example, in U.S. Pat. No. 5,981,200. It is understood thatgenetically encoded dyes such as GFP, BFP, CFP or YFP can form FRETpairs with each other, or can be combined with other appropriate donorfluorophores or acceptors. In one embodiment, the invention provides aclostridial toxin substrate in which the donor fluorophore and acceptorboth are genetically encoded. The desired toxin recognition sequence canbe engineered such that the cleavage site is between the chosen donorfluorophore/acceptor pair, and the substrate expressed, for example, inbacteria and purified.

In another embodiment, the invention provides a clostridial toxinsubstrate containing an acceptor which is a fluorophore with a longfluorescent lifetime of at least a microsecond. Such an acceptor, whichallows a time-resolved measurement of the fluorescence emission sincethe fluorescence lifetimes of impurities are generally in the nanosecondtimescale, can enhance the signal to noise ratio. A useful donorfluorophore/acceptor pair for time-resolved fluorescence can be, forexample, a europium cryptate donor fluorophore such as Eu-trisbipyridinecryptate (TBP-EU³⁺, λ_(Ex) 337 nm) combined with the 105 kDaphycobiliprotein acceptor fluorophore, allophycocyanin (Sittampalam etal., Curr. Opin. Chem. Biol. 1:384-391 (1997)). The Eu-trisbipyridinecryptate has two bipyridyl groups that harvest light and channel it tothe caged EU³⁺; this donor fluorophore has a long fluorescence lifetimeand nonradiatively transfers energy to allophycocyanin when in closeproximity to the acceptor, exhibiting greater than 50% transferefficiency at a donor fluorophore-acceptor distance of 9.5 nm. BothTBP-EU³⁺ and allophycocyanin and their spectroscopic characteristics arevery stable in biological media, and allophycocyanin emits (λ_(Em)=665nm) with the long lifetime of the donor, allowing time-resolveddetection (Kolb et al., J. Biomol. Screening 1:203-210 (1996)). Methodsof preparing substrates containing such donor fluorophore-acceptor pairsare well known in the art as described, for example, in Kolb et al.,supra, 1996, and Sittampalam et al., supra, 1997.

In a further embodiment, the invention relies on a non-fluorescentacceptor, sometimes designated a “true quencher.” A non-fluorescentacceptor can be useful, for example, in eliminating backgroundfluorescence resulting from direct (nonsensitized) acceptor excitation.A variety of non-fluorescent acceptors are known in the art including,for example, DABCYL and QSY® 7 dyes (see Molecular Probes, supra, 1996).

A clostridial toxin substrate of the invention contains a clostridialtoxin cleavage site which is positioned between a donor fluorophore andan acceptor. In one embodiment, the donor fluorophore is positionedamino-terminal of the cleavage site while the acceptor is positionedcarboxy-terminal of the cleavage site. In another embodiment, the donorfluorophore is positioned carboxy-terminal of the cleavage site whilethe acceptor is positioned amino-terminal of the cleavage site.

One skilled in the art understands that there are several considerationsin selecting and positioning a donor fluorophore and acceptor in aclostridial toxin substrate of the invention. The donor fluorophore andacceptor generally are positioned to minimize interference withsubstrate binding to, or proteolysis by, the clostridial toxin. Thus, adonor fluorophore and acceptor can be selected and positioned, forexample, so as to minimize the disruption of bonded and non-bondedinteractions that are important for binding, and to minimize sterichindrance. In addition, the spatial distance between the acceptor anddonor fluorophore generally is limited to achieve efficient energytransfer from the donor fluorophore to the acceptor.

As discussed above, efficiency of energy transfer from donor fluorophoreto acceptor is dependent, in part, on the spatial separation of thedonor fluorophore and acceptor molecules. As the distance between thedonor fluorophore and acceptor increases, there is less energy transferto the acceptor, and the donor fluorescence signal therefore increases,even prior to cleavage. The overall increase in fluorescence yield ofthe donor fluorophore, upon cleavage of the substrate, is dependent uponmany factors, including the separation distance between the donorfluorophore and acceptor in the substrate, the spectral overlap betweendonor fluorophore and acceptor, and the concentration of substrate usedin an assay. One skilled in the art understands that, as theconcentration of substrate increases, intermolecular quenching of thedonor, even after proteolytic cleavage, can become a factor. Thisphenomenon is denoted the “inner filter effect” (see below).

The Förster distance, which is the separation between a donorfluorophore and an acceptor for 50% energy transfer, represents aspatial separation between donor fluorophore and acceptor that providesa good sensitivity. For peptide substrates, adjacent residues areseparated by a distance of approximately 3.6 A in the most extendedconformation. For example, the calculated Förster distance for afluorescein/tetramethylrhodamine pair is 55A, which would represent aspatial separation between fluorescein and tetramethylrhodamine of about15 residues in the most extended conformation. Because peptides andpeptidomimetics in solution rarely have a fully extended conformation,donor fluorophores and acceptors can be more widely separated thanexpected based on a calculation performed using 3.6 A per residue andstill remain within the Förster distance.

Förster theory is based on very weak interactions between donorfluorophore and acceptor; spectroscopic properties such as absorption ofone fluorophore should not be altered in the presence of the other,defining the shortest distance range over which the theory is valid. Itis understood that, for many donor fluorophore-acceptor pairs, Förstertheory is valid when donor fluorophores and acceptors are separated byabout 10A to 100A. However, for particular donor fluorophore-acceptorpairs, Förster theory is valid below 10A as determined by subpicosecondtechniques (Kaschke and Ernsting, Ultrafast Phenomenon in Spectroscopy(Klose and Wilhelmi (Eds.)) Springer-Verlag, Berlin 1990.

Thus, in one embodiment, the invention provides a clostridial toxinsubstrate in which a donor fluorophore is separated from an acceptor bya distance of at most 100A. In other embodiments, the invention providesa clostridial toxin substrate in which a donor fluorophore is separatedfrom an acceptor by a distance of at most 90A, 80A, 70A, 60A, 50A, 40A,30A or 20A. In further embodiments, the invention provides a clostridialtoxin substrate in which a donor fluorophore is separated from anacceptor by a distance of 10A to 100A, 10A to 80A, 10A to 60A, 10A to40A, 10A to 20A, 20A to 100A, 20A to 80A, 20A to 60A, 20A to 40A, 40A to100A, 40A to 80A or 40A to 60A.

One skilled in the art understands that a clostridial toxin substrate ofthe invention can be designed to optimize the efficiency of FRET as wellas the ability to detect protease activity. One skilled in the artunderstands that a donor fluorophore can be selected, if desired, with ahigh quantum yield, and acceptor can be selected, if desired, with ahigh extinction coefficient to maximize the Förster distance. Oneskilled in the art further understands that fluorescence arising fromdirect excitation of an acceptor can be difficult to distinguish fromfluorescence resulting from resonance energy transfer. Thus, it isrecognized that a donor fluorophore and acceptor can be selected whichhave relatively little overlap of their excitation spectra such that thedonor can be excited at a wavelength that does not result in directexcitation of the acceptor. It further is recognized that a clostridialtoxin substrate of the invention can be designed so that the emissionspectra of the donor fluorophore and acceptor overlap relatively littlesuch that the two emissions can be readily distinguished. If desired, anacceptor having a high fluorescence quantum yield can be selected; suchan acceptor is preferred if acceptor fluorescence emission is to bedetected as the sole indicator of clostridial toxin protease activity,or as part of an emission ratio (see below).

It is understood that the donor fluorophore, acceptor, or both, can belocated within the active site cavity of botulinum or tetanus toxinholoenzyme. One skilled in the art understands that, if desired, aclostridial toxin substrate can be designed such that, when bound bytoxin, the donor fluorophore, acceptor, or both, is excluded from theactive site cavity of toxin holoenzyme. Thus, in one embodiment, theinvention provides a botulinum toxin substrate or tetanus toxinsubstrate in which, when bound by toxin, the donor fluorophore,acceptor, or both, is excluded from the active site cavity ofclostridial toxin holoenzyme. The invention provides, for example, aBoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F or BoNT/G substrate inwhich, when bound by toxin, the donor fluorophore, acceptor, or both, isexcluded from the active site cavity of toxin holoenzyme. In oneembodiment, the invention provides a BoNT/A substrate containing atleast six residues of human SNAP-25, where the six residues includeGln₁₉₇-Arg₁₉₈, in which the donor fluorophore, acceptor, or both, arenot positioned between residues Arg₁₉₁ to Met₂₀₂, which can be withinthe active site cavity of BoNT/A holoenzyme. In another embodiment, theinvention provides a BoNT/B substrate containing at least six residuesof VAMP-2, where the six residues include Gln₇₆-Phe₇₇, in which thedonor fluorophore, acceptor, or both, are not positioned betweenresidues Leu₇₀ to Ala₈₁ of VAMP-2, which are within the active sitecavity of BoNT/B holoenzyme.

In a complex of a VAMP substrate and the light chain of BoNT/B (LC/B),nearly all VAMP residues with side chains containing hydrogen bondacceptors or donors were hydrogen bonded with the LC/B. Thus, it isunderstood that a clostridial toxin substrate of the invention can beprepared, if desired, in which the potential for hydrogen bonding, forexample, by Ser, Thr, Tyr, Asp, Glu, Asn or Gln residues is notdiminished in the clostridial toxin substrate as compared to a nativeprotein sensitive to cleavage by the toxin. Thus, in particularembodiments, the present invention provides a clostridial toxinsubstrate in which the potential for hydrogen-bonding is not diminishedin the clostridial toxin substrate as compared to a native proteinsensitive to cleavage by the corresponding botulinum or tetanus toxin.

It is understood that, in addition to a donor fluorophore, acceptor andclostridial toxin recognition sequence, a clostridial toxin substrate ofthe invention can include, if desired, one or more additionalcomponents. As an example, a flexible spacer sequence such as GGGGS (SEQID NO: 84) can be included in a clostridial toxin substrate of theinvention. A substrate further also can include, without limitation, oneor more of the following: an affinity tag such as HIS6, biotin, or anepitope such as FLAG, hemagluttinin (HA), c-myc, or AU1; animmunoglobulin hinge region; an N-hydroxysuccinimide linker; a peptideor peptidomimetic hairpin turn; or a hydrophilic sequence, or anothercomponent or sequence that promotes the solubility or stability of theclostridial toxin substrate.

Methods for modifying proteins, peptides and peptidomimetics to containa donor fluorophore or acceptor are well known in the art (Faircloughand Cantor, Methods Enzymol. 48:347-379 (1978); Glaser et al., ChemicalModification of Proteins Elsevier Biochemical Press, Amsterdam (1975);Haugland, Excited States of Biopolymers (Steiner Ed.) pp. 29-58, PlenumPress, New York (1983); Means and Feeney, Bioconjugate Chem. 1:2-12(1990); Matthews et al., Methods Enzymol. 208:468-496 (1991); Lundblad,Chemical Reagents for Protein Modification 2nd Ed., CRC Press, BocaRatan, Fla. (1991); Haugland, supra, 1996). A variety of groups can beused to couple a donor fluorophore or acceptor, for example, to apeptide or peptidomimetic containing a clostridial toxin recognitionsequence. A thiol group, for example, can be used to couple a donorfluorophore or acceptor to the desired position in a peptide orpeptidomimetic to produce a clostridial toxin substrate of theinvention. Haloacetyl and maleimide labeling reagents also can be usedto couple donor fluorophores or acceptors in preparing a substrate ofthe invention (see, for example, Wu and Brand, supra, 1994.

Donor fluorophores and acceptors including proteins such as GFP andallophycocyanin (APC) can be attached to a clostridial toxin recognitionsequence by a variety of means. A donor fluorophore or acceptor can beattached by chemical means via a cross-linker moiety. Cross-linkers arewell known in the art, including homo- or hetero-bifunctionalcross-linkers such as BMH and SPDP. Where the donor fluorophore oracceptor is a protein, well known chemical methods for specificallylinking molecules to the amino- or carboxy-terminus of a protein can beemployed. See, for example, “Chemical Approaches to Protein Engineering”in Protein Engineering—A Practical Approach Rees et al. (Eds) OxfordUniversity Press, 1992.

One skilled in the art understands that contaminating substratescontaining only the donor fluorophore can result in high fluorescencebackground. Such background can be reduced or prevented, for example, byusing a relative excess of acceptor to donor fluorophore in preparationof the clostridial toxin substrate.

The present invention also provides kits for determining clostridialtoxin protease activity in a sample. The kit contains a clostridialtoxin substrate of the invention in a vial or other container. The kitgenerally also includes instructions for use. In one embodiment, a kitof the invention further includes as a positive control a known amountof the botulinum or tetanus toxin capable of cleaving the clostridialtoxin substrate included in the kit. In another embodiment, the kitcontains a clostridial toxin substrate of the invention and furtherincludes one or both cleavage products as a positive controls. In aparticular embodiment, the kit contains a clostridial toxin substrate ofthe invention and the corresponding cleavage product that includes thedonor fluorophore as a positive control. A kit of the inventionoptionally can include a container with buffer suitable for clostridialtoxin protease activity. A described further herein below, the methodsof the invention can be practiced with a combination of clostridialtoxin substrates. Thus, in one embodiment, the invention provides a kitfor determining clostridial toxin protease activity that includes atleast two clostridial toxin substrates of the invention.

The present invention also provides clostridial toxin targets useful fordetecting clostridial toxin protease activity. A clostridial toxintarget is a polypeptide, peptide or peptidomimetic which contains adonor fluorophore; an acceptor; and a clostridial toxin recognitionsequence that includes a cleavage site, where the cleavage siteintervenes between the donor fluorophore and the acceptor and where,under the appropriate conditions, energy transfer is exhibited betweenthe donor fluorophore and the acceptor. Energy can be transferred, forexample, via collisional energy transfer and does not require that theacceptor have an absorbance spectrum which overlaps the emissionspectrum of the donor fluorophore. Such a clostridial toxin target caninclude, for example, a botulinum toxin recognition sequence. Any of theclostridial toxin recognition sequences disclosed herein are useful in asubstrate of the invention also can be useful in a clostridial toxintarget of the invention. Selection and positioning of donor fluorophoresand acceptors such that collisional energy transfer is exhibited is wellknown in the art, as described, for example, in Gershkkovich andKholodovych, J. Biochem. Biophys. Methods 33:135-162 (1996).

The present invention also provides methods of determining clostridialtoxin protease activity. Such methods are valuable, in part, becausethey are amenable to rapid screening and do not require separation ofcleaved products from uncleaved substrate. Furthermore, the methods ofthe invention are applicable to crude samples as well as highly purifieddichain toxins and further are applicable to clostridial toxin lightchains, as described further below. The methods of the invention includethe following steps: (a) treating a sample, under conditions suitablefor clostridial toxin protease activity, with a clostridial toxinsubstrate that contains a donor fluorophore, an acceptor having anabsorbance spectrum overlapping the emission spectrum of the donorfluorophore, and a clostridial toxin recognition sequence containing acleavage site, where the cleavage site intervenes between the donorfluorophore and the acceptor and where, under the appropriateconditions, resonance energy transfer is exhibited between the donorfluorophore and the acceptor; (b) exciting the donor fluorophore; and(c) determining resonance energy transfer of the treated substraterelative to a control substrate, where a difference in resonance energytransfer of the treated substrate as compared to the control substrateis indicative of clostridial toxin protease activity. A method of theinvention can be practiced with an acceptor which is a fluorophore, orwith a non-fluorescent acceptor.

A method of the invention can be used to determine protease activity ofany clostridial toxin. In one embodiment, a method of the inventionrelies on a BoNT/A substrate to determine BoNT/A protease activity. ABoNT/A substrate useful in a method of the invention can be any of theBoNT/A substrates disclosed herein, for example, a BoNT/A substratecontaining at least six consecutive residues of SNAP-25, where the sixconsecutive residues include Gln-Arg. In another embodiment, a method ofthe invention relies on a BoNT/B substrate to determine BoNT/B proteaseactivity. A BoNT/B substrate useful in a method of the invention can beany of the BoNT/B substrates disclosed herein, for example, a BoNT/Bsubstrate containing at least six consecutive residues of VAMP, wherethe six consecutive residues include Gln-Phe. A method of the inventionalso can utilize a BoNT/C1 substrate to determine BoNT/C1 proteaseactivity. A BoNT/C1 substrate useful in a method of the invention can beany of the BoNT/C1 substrates disclosed herein, for example, a BoNT/C1substrate containing at least six consecutive residues of syntaxin,where the six consecutive residues include Lys-Ala, or containing atleast six consecutive residues of SNAP-25, where the six consecutiveresidues include Arg-Ala.

In another embodiment, a method of the invention relies on a BoNT/Dsubstrate to determine BoNT/D protease activity. A BoNT/D substrateuseful in a method of the invention can be any of the BoNT/D substratesdisclosed herein, for example, a BoNT/D substrate containing at leastsix consecutive residues of VAMP, where the six consecutive residuesinclude Lys-Leu. In a further embodiment, a method of the inventionrelies on a BoNT/E substrate to determine BoNT/E protease activity. ABoNT/E substrate useful in a method of the invention can be any of theBoNT/E substrates disclosed herein, for example, a BoNT/E substratecontaining at least six consecutive residues of SNAP-25, where the sixconsecutive residues include Arg-Ile. In yet a further embodiment, amethod of the invention relies on a BoNT/F substrate to determine BoNT/Fprotease activity. A BoNT/F substrate useful in a method of theinvention can be any of the BoNT/F substrates disclosed herein, forexample, a BoNT/F substrate containing at least six consecutive residuesof VAMP, where the six consecutive residues include Gln-Lys.

A method of the invention also can utilize a BoNT/G substrate todetermine BoNT/G protease activity. A BoNT/G substrate useful in amethod of the invention can be any of the BoNT/G substrates disclosedherein, for example, a BoNT/G substrate containing at least sixconsecutive residues of VAMP, where the six consecutive residues includeAla-Ala. A method of the invention also can be useful to determine TeNTprotease activity and, in this case, relies on a TeNT substrate. Any ofthe TeNT substrates disclosed herein can be useful in a method of theinvention, for example, a TeNT substrate containing at least sixconsecutive residues of VAMP, where the six consecutive residues includeGln-Phe.

A variety of samples are useful in the methods of the invention. Suchsamples include, but are not limited to, crude cell lysates; isolatedclostridial toxins; isolated clostridial toxin light chains; formulatedclostridial toxin products such as BOTOX-; and foodstuffs, includingraw, cooked, partially cooked and processed foods and beverages.

In a method of the invention, resonance energy transfer can bedetermined by a variety of means. In one embodiment, the step ofdetermining resonance energy transfer includes detecting donorfluorescence intensity of the treated substrate, where increased donorfluorescence intensity of the treated substrate as compared to thecontrol substrate is indicative of clostridial toxin protease activity.In another embodiment, the step of determining resonance energy transferincludes detecting acceptor fluorescence intensity of the treatedsubstrate, where decreased acceptor fluorescence intensity of thetreated substrate as compared to the control substrate is indicative ofclostridial toxin protease activity. In a further embodiment, the stepof determining resonance energy transfer includes detecting the acceptoremission maximum and the donor fluorophore emission maximum, where ashift in emission maxima from near an acceptor emission maximum to neara donor fluorophore emission maximum is indicative of clostridial toxinprotease activity. In an additional embodiment, the step of determiningresonance energy transfer includes detecting the ratio of fluorescenceamplitudes near an acceptor emission maximum to fluorescence amplitudesnear a donor fluorophore emission maximum, where a decreased ratio inthe treated sample as compared to the control sample is indicative ofclostridial toxin protease activity. In yet a further embodiment, thestep of determining resonance energy transfer is practiced by detectingthe excited state lifetime of the donor fluorophore in the treatedsubstrate, where an increased donor fluorophore excited state lifetimein the treated substrate as compared to the control substrate isindicative of clostridial toxin protease activity.

As discussed further below, a variety of conditions suitable forclostridial toxin protease activity are useful in a method of theinvention. For example, conditions suitable for clostridial toxinprotease activity can be provided such that at least 10% of thesubstrate is cleaved. Similarly, conditions suitable for clostridialtoxin protease activity can be provided such that at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 95% of the clostridial toxin substrateis cleaved, or such that 100% of the clostridial toxin substrate iscleaved. In one embodiment, the conditions suitable for clostridialtoxin protease activity are selected such that the assay is linear. Inanother embodiment, conditions suitable for clostridial toxin proteaseactivity are provided such that at least 90% of the clostridial toxinsubstrate is cleaved. In a further embodiment, conditions suitable forclostridial toxin protease activity are provided such that at most 25%of the clostridial toxin substrate is cleaved. In yet furtherembodiments, conditions suitable for clostridial toxin protease activityare provided such that at most 20%, at most 15%, at most 10% or at most5% of the clostridial toxin substrate is cleaved.

As used herein, the term “sample” means any biological matter thatcontains or potentially contains an active clostridial toxin, or lightchain or proteolytically active fragment thereof. Thus, the term sampleencompasses but is not limited to purified or partially purifiedclostridial toxin; recombinant single chain or dichain toxin with anaturally or non-naturally occurring sequence; chimeric toxin containingstructural elements from multiple clostridial toxin species or subtypes;recombinant toxin light chain with a naturally occurring ornon-naturally occurring sequence; bulk toxin; formulated product; cellsor crude, fractionated or partially purified cell lysates, for example,engineered to include a recombinant nucleic acid encoding a clostridialtoxin or light chain thereof, including bacterial, baculoviral and yeastlysates; raw, cooked, partially cooked or processed foods; beverages;animal feed; soil samples; water samples; pond sediments; lotions;cosmetics; and clinical formulations. It further is understood that theterm sample includes tissue samples, including, without limitation,mammalian samples, primate samples and human samples, and encompassingsamples such as intestinal samples, for example, infant intestinalsamples, and samples obtained from a wound. Thus, it is understood thata method of the invention can be useful, without limitation, to assayfor clostridial toxin protease activity in a food or beverage sample; toassay a sample from a human or animal, for example, exposed to aclostridial toxin or having one or more symptoms of a clostridial toxin;to follow activity during production and purification of clostridialtoxin, and to assay formulated clostridial toxin products, includingpharmaceuticals and cosmetics.

One skilled in the art understands that the methods of the invention aresuitable for assaying any protein or molecule with clostridial toxinprotease activity and do not rely, for example, on the ability of theclostridial toxin to bind to a neuronal cell or its ability to beinternalized or translocated across the membrane. Thus, the methods ofthe invention are suitable for assaying for proteolytic activity of aclostridial toxin light chain, alone, and, although useful for assayingsingle or dichain heterotoxin, do not require the presence of the heavychain. It further is understood that the methods of the invention areapplicable to non-neuronal clostridial toxins including native andrecombinant clostridial toxins, for example, clostridial toxinsengineered to target pancreatic acinar cells.

In the methods of the invention, a sample is treated with a clostridialtoxin substrate under conditions suitable for clostridial toxin proteaseactivity. Exemplary conditions suitable for clostridial toxin proteaseactivity are well known in the art, and further can be determined byroutine methods. See, for example, Hallis et al., J. Clin. Microbiol.34:1934-1938 (1996); Ekong et al., Microbiol. 143:3337-3347 (1997);Shone et al., WO 95/33850; Schmidt and Bostian, supra, 1995; Schmidt andBostian, supra, 1997; Schmidt et al., supra, 1998; and Schmidt andBostian, U.S. Pat. No. 5,965,699. It is understood that conditionssuitable for clostridial toxin protease activity can depend, in part, onthe specific clostridial toxin type or subtype being assayed and thepurity of the toxin preparation. Conditions suitable for clostridialtoxin protease activity generally include a buffer, such as HEPES, Trisor sodium phosphate, typically in the range of pH 5.5 to 9.5, forexample, in the range of pH 6.0 to 9.0, pH 6.5 to 8.5 or pH 7.0 to 8.0.Conditions suitable for clostridial toxin protease activity also caninclude, if desired, dithiothreitol, β-mercaptoethanol or anotherreducing agent, for example, where a dichain toxin is being assayed(Ekong et al., supra, 1997). In one embodiment, the conditions includeDTT in the range of 0.01 mM to 50 mM; in other embodiments, theconditions include DTT in the range of 0.1 mM to 20 mM, 1 to 20 mM, or 5to 10 mM. If desired, an isolated clostridial toxin or sample can bepre-incubated with a reducing agent, for example, with 10 mMdithiothreitol (DTT) for about 30 minutes prior to addition ofclostridial toxin substrate.

Clostridial toxins are zinc metalloproteases, and a source of zinc, suchas zinc chloride or zinc acetate, typically in the range of 1 to 500 μM,for example, 5 to 10 μM can be included, if desired, as part of theconditions suitable for clostridial toxin protease activity. One skilledin the art understands that zinc chelators such as EDTA generally areexcluded from a buffer for assaying clostridial toxin protease activity.

Conditions suitable for clostridial toxin protease activity also caninclude, if desired, bovine serum albumin (BSA). When included, BSAtypically is provided in the range of 0.1 mg/ml to 10 mg/ml. In oneembodiment, BSA is included at a concentration of 1 mg/ml. See, forexample, Schmidt and Bostian, supra, 1997.

The amount of clostridial toxin substrate can be varied in a method ofthe invention. Peptide substrate concentrations useful in a method ofthe invention include concentrations, for example, in the range of 5 μMto 3.0 mM. A peptide substrate can be supplied at a concentration, forexample, of 5 μM to 500 μM, 5 μM to 50 μM, 50 μM to 3.0 mM, 0.5 mM to3.0 mM, 0.5 mM to 2.0 mM, or 0.5 mM to 1.0 mM. The skilled artisanunderstands that the concentration of clostridial toxin substrate or theamount of sample can be limited, if desired, such that the assay islinear. At increasingly high concentrations of substrate or toxin,linearity of the assay is lost due to the “inner filter effect,” whichinvolves intermolecular energy transfer. Thus, in one embodiment, amethod of the invention relies on a clostridial toxin substrateconcentration which is limited such that intermolecular quenching doesnot occur. In another embodiment, a method of the invention relies on aclostridial toxin substrate concentration of less than 100 μM. Infurther embodiments, a method of the invention relies on a clostridialtoxin substrate concentration of less than 50 μM or less than 25 μM. Ifdesired, a linear assay also can be performed by mixing clostridialtoxin substrate with corresponding, “unlabeled” substrate which lacksthe donor fluorophore and acceptor of the clostridial toxin substrate.The appropriate dilution can be determined, for example, by preparingserial dilutions of clostridial toxin substrate in the correspondingunlabeled substrate.

The concentration of purified or partially purified clostridial toxinassayed in a method of the invention generally is in the range of about0.0001 to 5000 ng/ml toxin, for example, about 0.001 to 5000 ng/ml, 0.01to 5000 ng/ml, 0.1 to 5000 ng/ml, 1 to 5000 ng/ml, or 10 to 5000 ng/mltoxin, which can be, for example, purified recombinant light chain ordichain toxin or formulated clostridial toxin product containing humanserum albumin and excipients. Generally, the amount of purified toxinused in a method of the invention is in the range of 0.1 pg to 10 μg. Itis understood that purified, partially purified or crude samples can bediluted to within a convenient range for assaying for clostridial toxinprotease activity against a standard curve. Similarly, one skilled inthe art understands that a sample can be diluted, if desired, such thatthe assay for toxin protease activity is linear.

Conditions suitable for clostridial toxin protease activity alsogenerally include, for example, temperatures in the range of about 20°C. to about 45° C., for example, in the range of 25° C. to 40° C., orthe range of 35° C. to 39° C. Assay volumes often are in the range ofabout 5 to about 200 μl, for example, in the range of about 10 μl to 100μl or about 0.5 μl to 100 μl, although nanoliter reaction volumes alsocan be used with the methods of the invention. Assay volumes also canbe, for example, in the range of 100 μl to 2.0 ml or in the range of 0.5ml to 1.0 ml.

Assay times can be varied as appropriate by the skilled artisan andgenerally depend, in part, on the concentration, purity and activity ofthe clostridial toxin. In particular embodiments, at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the clostridial toxinsubstrate is cleaved. In further embodiments, the protease reaction isstopped before more than 5%, 10%, 15%, 20%, 25% or 50% of theclostridial toxin substrate is cleaved. Protease reactions can beterminated, for example, by addition of H₂SO₄ as in Example I, additionof about 0.5 to 1.0 sodium borate, pH 9.0 to 9.5, or addition of zincchelators. One skilled in the art understands that protease reactionscan be terminated prior to exciting the donor fluorophore or determiningenergy transfer.

As an example, conditions suitable for BoNT/A protease activity can beincubation at 37° C. in a buffer such as 30 mM HEPES (pH 7.3) containinga reducing agent such as 5 mM dithiothreitol; a source of zinc such as25 μM zinc chloride; and 1 μg/ml toxin (approximately 7 nM; Schmidt andBostian, supra, 1997). BSA in the range of 0.1 mg/ml to 10 mg/ml, forexample, 1 mg/ml BSA, also can be included when a sample is treated witha BoNT/A or other clostridial toxin substrate (Schmidt and Bostian,supra, 1997). If desired, BoNT/A, particularly dichain BoNT/A, can bepreincubated with dithiothreitol, for example, for 30 minutes beforeaddition of substrate. As another example, conditions suitable forclostridial toxin protease activity such as BoNT/A protease activity canbe incubation at 37° C. for 30 minutes in a buffer containing 50 mMHEPES (pH 7.4), 1% fetal bovine serum, 10 μM ZnCl₂ and 10 mM DTT with 10μM substrate (see Example I). As a further example, conditions suitablefor clostridial toxin protease activity, for example BoNT/B activity,can be incubation in 50 mM HEPES, pH 7.4, with 10 μM zinc chloride, 1%fetal bovine serum and 10 mM dithiothreitol, with incubation for 90minutes at 37° C. (Shone and Roberts, Eur. J. Biochem. 225:263-270(1994); Hallis et al., supra, 1996); or can be, for example, incubationin 40 mM sodium phosphate, pH 7.4, with 10 mM dithiothreitol, optionallyincluding 0.2% (v/v) Triton X-100, with incubation for 2 hours at 37° C.(Shone et al., supra, 1993). Conditions suitable for tetanus toxinprotease activity or other clostridial toxin protease activity can be,for example, incubation in 20 mM HEPES, pH 7.2, and 100 mM NaCl for 2hours at 37° C. with 25 μM peptide substrate (Cornille et al., supra,1994).

In a method of the invention for determining clostridial toxin proteaseactivity, a sample is treated with a clostridial toxin substrate thatcontains a first donor fluorophore, a first acceptor having anabsorbance spectrum which overlaps the emission spectrum of thedonorfluorophore, and a first clostridial toxin recognition sequencecontaining a cleavage site, where the cleavage site intervenes betweenthe donor fluorophore and the acceptor and where, under the appropriateconditions, resonance energy transfer is exhibited between the donorfluorophore and the acceptor. If desired, a second clostridial toxinsubstrate can be included; this second substrate contains a second donorfluorophore and second acceptor having an absorbance spectrum whichoverlaps the emission spectrum of the second donorfluorophore, and asecond clostridial toxin recognition sequence that is cleaved by adifferent clostridial toxin than the toxin that cleaves the firstclostridial toxin recognition sequence. The donor fluorophore-acceptorpair in the second substrate can be the same or different from thedonorfluorophore-acceptor pair in the first substrate. In this way, asingle sample can be assayed for the presence of multiple clostridialtoxins.

It is understood that one can assay for any combination of clostridialtoxins, for example, two, three, four, five, six, seven, eight, nine,ten or more clostridial toxins. One can assay, for example, anycombination of two, three, four, five, six, seven or eight of TeNT,BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G. For example,seven substrates, each containing fluorescein and tetramethylrhodamineflanking a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F or BoNT/Grecognition sequence can be treated with a sample under conditionssuitable for botulinum toxin protease activity before exciting the donorfluorescein at an absorption wavelength of about 488 nm and determiningenergy transfer. A shift in the emission maximum of the acceptor,tetramethylrhodamine (585 nm) to that of fluorescein (520 nm) isindicative of protease activity of at least one botulinum toxin. Such anassay can be useful, for example, for assaying food samples or tissuesamples for the presence of any clostridial toxin and can be combined,if desired, with one or more subsequent assays for individualclostridial toxins or specific combinations of clostridial toxins.

In another embodiment, a single sample is assayed for two or moredifferent clostridial toxins using two or more different clostridialtoxin substrates with each substrate containing a different donorfluorophore-acceptor pair. The use of multiple substrates can be usefulfor extending the dynamic range of the assay, as described, for example,in U.S. Pat. No. 6,180,340. As an example of the use of multipleclostridial toxin substrates, a single sample can be assayed for BoNT/Aand BoNT/B protease activity using a first clostridial toxin substratecontaining the donor fluorophore fluorescein and the acceptortetramethylrhodamine with an intervening BoNT/A recognition sequence,and a second clostridial toxin substrate containing the donorfluorophore EDANS and the acceptor DABCYL with an intervening BoNT/Brecognition sequence. The first donor fluorophore, fluorescein, isexcited at about 488 nm, and energy transfer is determined, withincreased first donor fluorescence intensity at about 520 nm indicativeof BoNT/A protease activity. The second donor fluorophore, EDANS, isexcited at an absorption wavelength of about 340 nm, with increasedsecond donor fluorescence intensity (490 nm) indicative of BoNT/Bprotease activity. Similarly, where two or more different donorfluorophores are to be used together to assay a single sample, one cancombine, for example, any combination or all of the followinglanthanides: terbium, dysprosium, europium and samarium (EG&G-Wallac).These lanthanides have spectra that are clearly distinguishable on thebasis of decay time and wavelength. Those skilled in the art understandthat the first donor fluorophore can be excited before, at the sametime, or after excitation of the second donor fluorophore, and thatenergy transfer of the first substrate can be determined before, at thesame time, or after determining energy transfer of the second substrate.

Multiple substrates also can be used in the methods of the invention toextend the range of the assay. In one embodiment, at least twoclostridial substrate are used together at different dilutions; thesubstrates have donor fluorophore-acceptor pairs and, therefore, areseparately detectable, but have recognition sequences for the sameclostridial toxin. In another embodiment, otherwise identicalclostridial toxin substrates with different donor fluorophore-acceptorpairs are used together at different dilutions to extend the range ofthe assay.

The methods of the invention involve exciting the donor fluorophorecontained in the clostridial toxin substrate. One skilled in the artunderstands that a donor fluorophore generally is excited at or near theoptimal absorption wavelength (excitation wavelength) of the donorfluorophore. Where the donor fluorophore is fluorescein, the donor canbe excited, for example, at or near the optimal absorption wavelength of488 nm.

Proteolysis of the clostridial toxin substrate, and hence clostridialtoxin protease activity, can be detected by a variety of means, forexample, by detecting an increased donor fluorescence intensity; adecreased acceptor fluorescence intensity; a shift in emission maximafrom near the acceptor emission maximum to near the donor fluorophoreemission maximum; a decreased ratio of fluorescence amplitudes near theacceptor emission maximum to the fluorescence amplitudes near the donorfluorophore emission maximum; or an increased donor fluorophore excitedstate lifetime. It is understood that the relevant fluorescenceintensities or excited state lifetimes are detected at the appropriateselected wavelength or range of wavelengths. For example, where donorfluorescence intensity is detected, the appropriate selected wavelengthat or near the emission maxima of the donor fluorophore, or a range ofwavelengths encompassing or near to the emission maxima of the donorfluorophore.

It is recognized that changes in the absolute amount of substrate,excitation intensity, and turbidity or other background absorbance inthe sample at the excitation wavelength effect the fluorescenceintensities of donor and acceptor fluorophores roughly in parallel.Thus, it is understood that a ratio of emission intensities isindependent of the absolute amount of substrate, excitation intensity,or turbidity or other background absorbance, and can be a usefulindicator of clostridial toxin protease activity. Similarly, one skilledin the art understands that the excitation state lifetime of a donorfluorophore is independent of the absolute amount of substrate,excitation intensity, or turbidity or other background absorbance andcan be useful in a method of the invention.

In one embodiment, donor fluorescence intensity is detected, withincreased donor fluorescence intensity indicative of clostridial toxinprotease activity. Such increased intensity can be, for example, atleast two-fold, three-fold, five-fold, ten-fold, twenty-fold or morerelative to fluorescence intensity at the same wavelength of the sameclostridial toxin substrate not contacted with sample.

For detection of donor fluorescence intensity, excitation is set at thewavelength of donor fluorophore absorption, and the emission of thedonor fluorophore is monitored. The emission wavelength of the donorfluorophore generally is selected such that little or no contributionfrom acceptor fluorescence is observed. The presence of acceptorquenches donor fluorescence. Energy transfer efficiency, E, iscalculated from E=1−I_(DA)/I_(D), where I_(DA) and I_(D) are donorintensities in the presence and absence of acceptor. Both are normalizedto the same donor fluorophore concentration. If desired, time resolvedmeasurements, for which donor fluorophore concentration is not required,can be performed, E=1−{T_(DA)}/T_(D), where {T_(DA)} and {T_(D)} areamplitude-averaged lifetimes of donor fluorophore in the presence andabsence of acceptor.

In one embodiment, a shift in emission maxima from near the acceptoremission maximum to near the donor fluorophore emission maximum isdetected as a determination of resonance energy transfer. Where atetramethylrhodamine acceptor is combined with the donor fluorophorefluorescein, one can detect a shift from predominantly red emission topredominantly green emission as an indicator of decreased resonanceenergy transfer and, therefore, of clostridial toxin protease activity.It is understood that the observed shift in emission maxima generallywill not be a complete shift but that only part of the emissionintensity will be shifted to near the donor fluorophore emissionmaximum.

In the methods of the invention, resonance energy transfer of thetreated substrate is determined relative to a control substrate. Such acontrol substrate generally can be, for example, the same clostridialtoxin substrate which is not treated with any sample, or which istreated with a defined sample containing one or more clostridial toxin.One skilled in the art understands that a variety of control substratesare useful in the methods of the invention and that a control substratecan be a positive control substrate or a negative control substrate. Acontrol substrate can be, for example, a negative control such as asimilar or identical substrate that is contacted with a similar samplethat does not contain active clostridial toxin, or that is not contactedwith any sample. A control substrate also can be, for example, apositive control such as the two purified cleavage products that resultfrom clostridial toxin proteolysis of the clostridial toxin substrate. Acontrol substrate can be the donor fluorophore-containing cleavageproduct, the acceptor-containing cleavage product, or a combination ofboth.

The methods of the invention for determining clostridial toxin proteaseactivity involve determining resonance energy transfer of a clostridialtoxin substrate treated with a sample relative to a control substrateand can be practiced as “fixed-time” assays or as continuous timeassays. Thus, in one embodiment, the FRET determination is repeated atone or more later time intervals. Fluorescence resonance energy transfercan be determined, for example, at two or more, five or more, ten ormore, or twenty or more different intervals. Fluorescence intensitiesand other indicators of FRET also can be detected continuously by wellknown methods (see, for example, Wang et al., supra, 1993; Holskin etal., supra, 1995; and Kakiuchi et al., supra, 1999).

In a method of the invention, fluorescence of a treated substrate isdetermined using a fluorimeter. In general, excitation radiation from anexcitation source having a first wavelength passes through excitationoptics. The excitation optics cause the excitation radiation to excitethe substrate. In response, fluorophores in the substrate emit radiationwhich has a wavelength that is different from the excitation wavelength.Collection optics then collect the emission; if desired, the deviceincludes a temperature controller to maintain the clostridial toxinsubstrate at a specific temperature while being scanned. If desired, amulti-axis translation stage moves a microtiter plate containing aplurality of samples in order to position different wells to be exposed.It is understood that the multi-axis translation stage, temperaturecontroller, auto-focusing feature, and electronics associated withimaging and data collection can be managed by the appropriate digitalcomputer.

Thus, the methods of the invention can be automated and, furthermore,can be configured in a high-throughput or ultra high-throughput formatusing, for example, 96-well, 384-well or 1536-well plates. As oneexample, fluorescence emission can be detected using Molecular DevicesFLIPR—instrumentation system (Molecular Devices; Sunnyvale, Calif.),which is designed for 96-well plate assays (Schroeder et al., J. Biomol.Screening 1:75-80 (1996)). FLIPR utilizes a water-cooled 488 nm argonion laser (5 watt) or a xenon arc lamp and a semiconfocal optimal systemwith a charge-coupled device (CCD) camera to illuminate and image theentire plate. The FPM-2 96-well plate reader (Folley Consulting andResearch; Round Lake, Ill.) also can be useful in detecting fluorescenceemission in the methods of the invention. One skilled in the artunderstands that these and other automated systems with the appropriatespectroscopic compatibility such as the ECLIPSE cuvette reader(Varian-Cary; Walnut Creek, Calif.), the SPECTRA_(max) GEMINI XS(Molecular Devices) and other systems from, for example, from PerkinElmer can be useful in the methods of the invention.

The following examples are intended to illustrate but not limit thepresent invention.

Example I Analysis of BoNT/A Activity Using Fluorescence ResonanceEnergy Transfer

This example describes the use of a FRET assay to analyze proteolyticactivity of a botulinum toxin.

The FRET substrateX1-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Z2-NH₂(SEQID NO: 85) was synthesized by Alpha Diagnostics International (SanAntonio, Tex.). This substrate contains a recognition sequence forBoNT/A flanked by a fluorescein-modified lysine residue (“X1”) and atetramethylrhodamine-modified lysine residue (“Z2”) followed by acarboxy-terminal amide. Following proteolysis by botulinum toxinserotype A, the cleavage productsX1-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln (SEQ ID NO: 86) andArg-Ala-Thr-Lys-Met-Leu-Z2-NH₂ (SEQ ID NO: 87) are produced.

Additional FRET substrates also are synthesized:X1-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-Z2—NH₂(SEQ ID NO: 88);X1-Ala-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Z2-NH₂(SEQ ID NO: 89);X1-Ala-Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-Z2—NH₂(SEQ ID NO: 90);X1-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Z2-NH₂ (SEQID NO: 91);X1-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-Z2—NH₂(SEQ ID NO: 92);X1-Met-Glu-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-Z2-NH₂(SEQID NO: 93), in each of which X1 is a fluorescein-modified lysine residueand Z2 is a tetramethylrhodamine-modified lysine residue;X3-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Z4-NH₂ (SEQID NO: 94), in which X3 is a DABCYL modified lysine residue and Z4 is aEDANS modified glutamate residue; andX3-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu-Gly-Ser-Gly-Z5-NH₂(SEQ ID NO: 95), in which X3 is a DABCYL modified lysine residue and Z5is a EDANS modified lysine residue.

Purified BoNT/A light chain (LC/A) or cellular extract containing LC/Ais diluted in assay buffer (0.05 M HEPES (pH 7.4); 1% FBS; 10 μM ZnCl₂;and 10 mM DTT). Dichain BoNT/A is incubated with 10 mM dithiothreitol(DTT) for about 30 minutes prior to analysis. Reactions contain variousconcentrations of LC/A, dichain toxin or formulated BOTOX-product, from0.1 ng to 10 μg. Toxin is assayed as follows: FRET substrate is added toa final concentration of 10 μM in a final volume of 100 μL assay buffer.The reaction is incubated at 37° C. for 30 minutes, and is subsequentlyterminated by addition of 50 μL 2M H₂SO₄.

Fluorescence is measured in a fluorimeter microplate reader (MolecularDevices SPECTRA_(max) GEMINI XS) with λ_(ex)=488 nM, λ_(em)=520 nM andλ_(em)=585 nm. A reduction of at least about 5% in the λ_(em)=585 nm isindicative of BoNT/A protease activity. An increase of about 5% in theλ_(em)=520 nm also is indicative of BoNT/A protease activity of thedichain or light chain botulinum toxin.

Kinetic assays are performed as follows. Several reactions containingthe same amount of LC/A or dichain toxin are initiated in the buffer andunder the conditions described above. Different reactions are thenstopped at two or five minute intervals, and fluorescence detected asdescribed above.

These results demonstrate that botulinum toxin proteolytic activity canbe assayed with an intramolecularly quenched FRET substrate.

All journal article, reference and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1. A Clostridial toxin substrate, comprising: a) a donor fluorophore; b)an acceptor having an absorbance spectrum overlapping the emissionspectrum of said donor fluorophore; and c) a Clostridial toxinrecognition sequence comprising a Clostridial toxinP5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence, saidClostridial toxin P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage sitesequence intervening between said donor fluorophore and said acceptor;wherein either of said donor fluorophore, said acceptor, or both saiddonor fluorophore and said acceptor are genetically encoded; andwherein, under the appropriate conditions, resonance energy transfer isexhibited between said donor fluorophore and said acceptor.
 2. Thesubstrate of claim 1, wherein said donor fluorophore is geneticallyencoded.
 3. The substrate of claim 1, wherein said acceptor isgenetically encoded.
 4. The substrate of claim 1, wherein said donorfluorophore and said acceptor are genetically encoded.
 5. The substrateof claim 1, wherein said donor fluorophore and said acceptor areseparated by at most forty residues, at most thirty residues, at mosttwenty residues, at most fifteen residues, at most ten residues, at mosteight residues, or at most six residues.
 6. The substrate of claim 1,wherein said substrate can be cleaved with an activity of at least 1nanomoles/minute/milligram toxin, at least 20 nanomoles/minute/milligramtoxin, at least 50 nanomoles/minute/milligram toxin, at least 100nanomoles/minute/milligram toxin, or at least 150nanomoles/minute/milligram toxin.